Unit 1: Bio/Biochem
Enzymes in which oxygen is the final electron acceptor often include what in their names?
"oxidase"
What does it mean when the DNA double helix is called *Z-DNA*?
"zig-zag" less common form: *left*-handed helix with turns every *4.6 nm* and contains *12* bases within each turn (see how there's more space in b/w each turn and thus more bases per turn)
How are terpenes grouped?
# isoprene units
Define DNA polymerase *processivity*.
# of nucleotides that a polymerase can incorporate into DNA during a single template‐binding event, before dissociating from a DNA template. ✴︎The overall efficiency of DNA synthesis increases when the processivity of a polymerase increases.
What is *kcat*?
# of substrate molecules "turned over", converted to product, per enzyme molecule per second
Usually, chiral *L-amino acids* display the (___) absolute configuration, with the only exception being cysteine.
(S) *cytsteine is still an L-amino acid, but it has the (R) absolute configuration.
What are the 3 types of *wobble* pairing proposed thus far?
(i) U in the wobble position of the tRNA anticodon pairs with A or G of codon (ii) G pairs with U or C (iii) I pairs with A, U or C.
Proofreading always occurs in what direction?
*3' ⟶ 5'*
What happens during *DNA methylation*?
*DNA methylases* add methyl groups to C and A nucleotides Often *silences* genes This process is important during *development* as it functions to silence genes that no longer need to be activated Being heavily methylated, heterochromatin is literally blocked off to any access by transcriptional machinery.
What is the different between *genomic* DNA libraries and *cDNA* libraries?
*Genomic* ￫ contain large fragments of DNA and include both coding (exon) and noncoding (intron) regions of the genome *cDNA (expression)* ￫ constructed by reverse-transcribing processed mRNA, thus lacks noncoding (intron) regions. Only includes genes that are expressed in the tissue from which the mRNA was isolated!
Describe the *phospholipase C* cascade that increases intracellular calcium levels.
*Gq* stimulates ⇝ *phospholipase C* *phospholipase C* cleaves a phospholipid from the membrane, forming *PIP2* *PIP2* is cleaved ⥤ ① *DAG* and ② *IP3* *IP3* opens Ca²⁺ channels in the ER, ↑ intracellular Ca²⁺
What are the 3 main types of *G proteins*?
*Gs* - *s*timulates adenylate cyclase, which increases cAMP levels in the cell *Gi*- *i*nhibits adenylate cyclase, which decreases cAMP levels in the cell *Gq* - activates *phospholipase C*, which cleaves a phospholipid to form PIP2, which is cleaved into IP3 and DAG. IP3 can open calcium channels and increase cellular levels.
During which phases of the cell cycle are *nucleotide & base excision repair* mechanisms active?
*G₁* and *G₂*
Kinesins and dyneins have *opposite* polarities. Which does which?
*Kinesins* bring vesicles toward the *positive* end of the microtubule *Dyneins* bring vesicles toward the *negative* end of a microtubule
Distinguish *nucleosome* from *chromatosome*.
*Nucleosome* ￫ DNA wrapped around 8 histone proteins *Chromatosome* ￫ nucleosome + H1 + linker DNA
Which protein is needed by both DNA polymerase *δ* and *ε*?
*PCNA* ￫ assembles into a trimer to form the *sliding clamp* which strengthens the interaction b/w these polymerases and the template strand.
What is the difference between *promoters* and *enhancers*?
*Promoters* ￫ within 25 bp of the transcription start site *Enhancers* ￫ more than 25 bp of the transcription start site
During which phases of the cell cycle are *mismatch repair* mechanisms active?
*S* (proofreading) and *G₂* (MSH2 and MLH1)
Describe the structure of an *operon*
*Structural gene* ￫ codes for the protein further upstream ⟵ *Operator* ￫ nontranscribable region that is capable of binding a *repressor* further upstream ⟵⟵ *Promoter* ￫ similar to the promoter in eukaryotes, it allows RNA polymerase to bind further upstream ⟵⟵⟵ *Regulator gene* ￫ codes for the *repressor* Another component that is not *on* the operator, but functions to bind the repressor and thereby facilitate transcription is the *inducer* a small protein that can bind the inducer, preventing its binding to the operator.
In the context of *cooperativity*, binding of the substrate encourages the transition of other subunits from which state to which state?
*T* state ⟶ *R* state
What happens during *histone acetylation*?
*TF*s bind to DNA and recruit other coactivators, like *histone acetylases* These enzymes acetylate *lysine* residues found in the amino terminal tail region of the histones. This *decreases* the positive charge on LYS residues and *weakens* the interaction of the histone w/ DNA Which allows for easier access to the DNA ⟶ transcription!
Show the reaction mechanism for *aminoacyl-tRNA synthetase* in activation of AA for protein synthesis.
*aminoacyl-tRNA synthetase* (unique type for each amino acid, so 20) transfers the *activated* amino acid to the 3' end of the tRNA using *two* high-energy bonds from ATP.
How is *cytosine deamination* repaired?
*base excision repair* Details: U is detected by glycosylase since it is not normal in DNA, and it is excised and replaced with cytosine
Non-helix distorting mutations are repaired by...
*base-excision repair* If mutations *do* distort the helix (i.e. thymine dimers), *nucleotide* excision repair is required.
DNA unwinds at origins of replication and then the generation of new DNA proceeds in what direction?
*both*, creating *replication forks* ✴︎increased efficiency
When amino acids have acidic side chains, which groups are used to calculate the isoelectric point?
*carboxyl group* + *side chain* / 2 ✴︎Ignore the amino group!
Whenever substrate concentration is increasing (decreased affinity, so higher Km), where is the Lineweaver Burk plot moving?
*closer* to the origin, where [S] is *infinite*!
Which region on an *antibody* that is involved in recruitment and binding of other cells of the immune system?
*constant* region
Which type of chromatin can be transcribed?
*euchromatin* Explanation: it's *relaxed* and unwound, so it can be accessed. Heterochromatin is tightly packed and therefore silent
What are the key genes/gene products & enzymes involved in *base excision repair*?
*eukaryotes*: *glycosylase, AP endonuclease*, DNA polymerase β (short-patch) or DNA polymerases δ & ε (long-patch), DNA ligase
What specific genes encode the enzymes that function in G₂ phase *mismatch repair*?
*eukaryotes*: MSH2 and MLH1 *prokaryotes*: MutS and MutL
Breaking the bonds of *ATP* is an (endothermic/exothermic) process?
*exo*thermic ★This is an important exception to most other bond breaking, which requires an input of energy (*endo*thermic). This is exothermic because the - phosphate groups are so close & repel each other quite strongly to the point that removing the terminal phosphate group releases energy *this is what powers our cells*
The 3 main groups of *ion channels* have different mechanisms of opening; however, they are all alike in that they permit ____________ of charged particles.
*facilitated diffusion*
phosphatidylcholine (PC)
*glycerophospholipid* w/ a choline head group
phosphatidylethanolamine (PE)
*glycerophospholipid* w/ an ethanolamine head group
How do alterations to *chromatin* structure contribute to control of gene expression in eukaryotes?
*heterochromatin* is *not* accessible to transcriptional machinery, so the genes it contains are inactive So it must be transformed into *euchromatin* Two Ways to Change Chromatin: ① *Histone acetylation* weakens the interaction of the histone w/ DNA, thus allowing for an open (accessible) conformation. The opposite process (histone *de*acetylation) results in gene silencing ② DNA methylation often *silences* gene expression as heterochromatin regions of DNA are much more heavily methylated (steric hindrance!) hindering access of transcriptional machinery to the DNA.
Neurotransmitters act as what type of ion channels at the postsynaptic membrane?
*ligand-gated* (they are the ligand)
If a strand of RNA contained 15% cytosine, 15% adenine, 35% guanine, and 35% uracil, would this violate Chargaff's rules? Why or why not?
*no* Explanation: the question says *RNA* which is single-stranded and does not adhere to the complementarity seen in DNA. %C does not necessarily equal %G and %A does not necessarily equal %U. *Chargaff's rules apply to DNA*
What type of biological activity can be attributed to *Z-DNA*?
*none* Explanation: unstable and difficult to research
Where is *proline* usually found in the secondary structure of proteins?
*not* in the middle of either α-helices or β-pleated sheets, but in the terms between the chains of a β-pleated sheet (*turns*) and as the residue at the *start* of an α-helix (*kinks*)
What type of adhesion do *selectins* facilitate?
*one* cell to *carbohydrates* usually on surface of *other* cells
What type of adhesion do *integrins* facilitate?
*one* cell to *proteins* in the extracellular matrix (ECM)
When denaturing DNA for replication and transcription, what bonds are broken?
*only* hydrogen bonds that allow for base pairing the covalent bonds b/w nucleotides in the backbone of the DNA *remain* (otherwise, you wouldn't even have a single strand, it would just be a bunch of nucleotides floating around)
Where does RNA polymerase III synthesize the 5S rRNA?
*outside* the nucleolus
How does *negative feedback* work with regards to enzyme activity?
*product* may bind to the active site of an enzyme or multiple enzymes that acted earlier in its biosynthetic pathway, thereby *competitively inhibiting* these enzymes and making them unavailable for use
What are the key genes/gene products & enzymes involved in *mismatch repair*?
*prokaryotes*: - genes: MutS and MutL; PCNA (protein) - enzymes: variable exonuclease, DNA polymerase III, DNA ligase, DNA methylase *eukaryotes*: - genes: MSH2 and MLH1; PCNA (protein), RFC (protein) - enzymes/proteins: variable exonuclease, DNA polymerase δ, DNA ligase, DNA methylase
Name the corresponding enzyme that *removes positive supercoiling by introducing nicks in the DNA strand* ahead of advancing replication forks for both prokaryotes and eukaryotes.
*prokaryotes*: DNA topoisomerases (Type I) *and* DNA gyrase (a/k/a Type II isomerase) *eukaryotes*: DNA topoisomerases (Type I)
What are the key genes/gene products & enzymes involved in *nucleotide excision repair*?
*prokaryotes*: helicase, various *excision endonucleases*, DNA polymerase I, DNA ligase *eukaryotes*: helicase, various *excision endonucleases*, DNA polymerase δ, ε, and/or κ, DNA ligase
What is the role of *DNA polymerase β*?
*repair* Details: esp. *base excision repair*, specifically "short-patch" Mnemonic: *b*eta ￫ *b*ase excision
What is the role of *DNA polymerase δ*?
*replication and fills in gaps* ✴︎Details: ① *5' ⟶ 3' polymerase*: involved in primary synthesis along with α & ε, but δ is known for its activity on the *lagging strand* as it functions to fill in gaps left behind when RNA primers are removed with new DNA nucleotides. ② *3' ⟶ 5'* exonuclease activity (proofreading)
What is the role of *DNA polymerase ε*?
*replication and repair* ✴︎Details: ① *5' ⟶ 3' polymerase*: also involved in synthesis on both leading & lagging strands along with α & δ, but esp on the *leading strand*. It is deeply involved in DNA repair via *nucleotide* and *base excision repair* ② some say *3' ⟶ 5'* exonuclease activity (proofreading)
When amino acids have basic side chains, which groups are used to calculate the isoelectric point?
*side chain* + *amino group* / 2 ✴︎Ignore the carboxyl group!
What factor is required by prokaryotic *RNA Polymerase* for efficient binding and transcription?
*sigma factor* different sigma factors recognize different promoter sequences RNA Polymerase + σ factor = RNA Polymerase *holoenzyme* It is analogous to the eukaryotic *TFIIB*
What is the key to reannealing ssDNA?
*slowly* removed the denaturing condition Ex: *slowly* cooling, *slowly* decreasing the pH from alkaline ⟶ neutral, etc.
When is using *genomic* DNA libraries useful?
*studying* introns, centromeres, telomeres, etc. because these are *noncoding* regions ❗️can *not* use to express recombinant proteins or perform gene therapy
How do *solutes* such as urea and detergents denature proteins?
*they disrupt elements of 2°, 3° and 4° structure* *Urea*: directly interferes with forces that hold proteins together Ex: • disrupt 3° and 4° structure by breaking disulfide bridges • overcome hydrogen bonds and other side chain interactions (breaking 2° structure) *Detergents (like SDS)*: solubilize proteins, disrupting noncovalent hydrophobic bonds and promoting denaturation ❗️Detergents will NOT disrupt covalent bonds (i.e. disulfide bridges) unless it specifies *reducing conditions* ✷ DDT and/or β-mercaptoethanol CAN reduce disulfide bonds
What are prosthetic groups?
*tightly* bound cofactors or coenzymes that are necessary for enzyme function They have their own distinct name because some cofactors and coenzymes are only bound by weak noncovalent interactions, so prosthetic groups are way more crucial to the enzyme.
What type of adhesion do *cadherins* facilitate?
*two* cells of same/similar type using *calcium*
free fatty acids
*unesterified* fatty acids with a free carboxylate group
prostaglandins
*unsaturated* fatty acids derived from *arachidonic acid* and contain 1 five-carbon ring act as *paracrine* or autocrine* signalers
What type of channels serve as the pacemaker current in cells of the sinoatrial node?
*voltage-gated* (nonspecific) sodium-potassium channels
The anode is what charge in *isoelectric focusing*?
+
The site on the DNA from which the first RNA nucleotide is transcribed is called the _____________.
+1 or *initiation* site
Why is the overall charge on lysine in its fully protonated state (low pH)?
+2 Explanation: 2 amino groups (both +1) and 1 carboxyl (neutral when protonated)
If the pH is greater than the pI, the protein will be (+/-) charged.
-
What mechanisms are involved in *posttranscriptional processing* to the pre-mRNA before it exits the nucleus?
- addition of poly-A tail - splicing out of introns and other things
Signaling role of lipids
- coenzymes in ETC & glycosylation - hormones - intracellular messengers - light-absorbing molecules - act as pigments in plants & animals
What are the *safety* concerns regarding DNA technology?
- increased resistance in viruses and bacteria can impact both humans and the environment
What are some examples of *covalent modifications* to enzymes?
- phosphorylation - glycosylation
What are the elements comprising a phospholipid?
- polar head: phosphate + alcohol linked to - backbone by phosphodiester linkages - nonpolar tail: fatty acids linked to backbone by ester linkages
Where are adipocytes primarily found?
- under skin - around mammary glands - abdominal cavity
When _______ equivalents of base have been added to solution, the pH equals pKa₁.
0.5 *[fully protonated form] = [zwitterion] ✴︎flat region (first blue box)
*Pyrimidines* contain how many rings?
1
How many heads does *myosin* have?
1 only one neck too
biologically active form of vitamin D
1,25-dihydroxyvitamin D [1,25(OH)2D] or calcitriol
How is *glycogen* broken down?
1. Glycogen phosphorylase path (skeletal muscle) ￫ cleaves from nonreducing (acetal) end of a glycogen branch and then phosphorylates it, producing glucose 1-phosphate 2. Lysosomal breakdown (alpha-1-4-glucosidase)
When we've added _______ equivalents of base, the pH equals the isoelectric point (pI) of the amino acid.
1.0 *every molecule is a zwitterion at this point
When ______ equivalents of base have been added, the pH equals the pKa₂.
1.5 *second buffering phase where [zwitterion] = [fully deprotonated form] ✴︎flat region (second blue box)
What is the y-intercept on a *Lineweaver Burke* plot?
1/Vmax occurs when x = 0 (1/[S] = 0) therefore [S] is infinite
The rate of a reaction doubles every ____°C up to a certain point (37°C in the body).
10
Convert daltons to g/mol.
100 daltons = 100 g/mol ⁂ this is the average molar mass of one amino acid
*Purines* contain how many rings?
2
Adenine (A) always pairs with thymine (T) via ____ hydrogen bonds.
2
A single terpene contains how many isoprene units?
2 monoterpenes (C₁₀H₁₆)
*Integrins* have how many membrane-spanning chains?
2 (*α* and *β*)
How many heads do *kinesins* and *dyneins* have?
2, with at least one staying attached to tubulin *at all times*
When _______ equivalents of base are added, the amino acid has become fully deprotonated.
2.0
How many carbons do prostaglandins have?
20
How many carbons are in a diterpene?
20 Explanation: One terpene unit is made from two isoprene units, each of which has five carbons.
Using just the 20 common amino acids, how many total tripeptides can be created?
20 × 20 × 20 = 8,000
Antibodies have ____ heavy chain/s and ____ light chain/s.
2; 2
Forming a disulfide bond requires the loss of _____ proton/s and ______ electron/s.
2; 2
Guanine (G) always pairs with cytosine (C) via ____ hydrogen bonds.
3
Which of the *D* stereoisomers for *D-glucose* are epimers of D-glucose?
3 D-mannose (C2) D-allose (C3) D-galactose (C4) Read carefully, it says *D* stereoisomers, so even though L-idose is the C-5 epimer of D-glucose, since it is not itself a D stereoisomer, it does not answer the question. And D-idose differs from glucose at 3 chiral centers, so it cannot be considered an epimer, though it is a D stereoisomer for D-glucose
Triose
3 carbon sugar
*RNA polymerase II* builds an RNA strand in the 5' to 3' direction, adding each new nucleotide to the ___ end of the strand.
3'
Polymerases can only add nucleotides to what end of a new DNA strand?
3' Explanation: Remember, it *synthesizes from 5' ⟶ 3', so naturally it keeps adding onto the *end* which would be the 3'. They use the free -OH group found at the 3' end as a "hook," adding a nucleotide to this group in the polymerization reaction. http://scienceprimer.com/dna-polymerase
In *phosphodiester bonds*, the ____ end of one nucleotide's sugar joins the ____ end of the adjacent nucleotide's sugar.
3' 5'
In what direction does *DNA polymerase* read the template strand?
3' ⟶ 5' so that it can synthesize the complementary strand in the 5' ⟶ 3'
How many strands do *eukaryotic* rRNA have?
4 28S 18S 5.8S 5S
If chymotrypsin cleaves at the carboxyl end of phenylalanine, tryptophan, and tyrosine, how many oligopeptides would be formed in enzymatic cleavage of the following molecule with chymotrypsin? Val-Phe-Glu-Lys-Tyr-Phe-Trp-Ile-Met-Tyr-Gly-Ala
4 Explanation: Val-Phe/Glu-Lys-Tyr/Phe/Trp/Ile-Met-Tyr/Gly-Ala ⟶ Val-Phe Glu-Lys-Tyr Ile-Met-Tyr Gly-Ala ✴︎Phe and Trp are left on their own and so would *not* be considered oligopeptides (or any kind of peptide for that matter)
Tetrose
4 carbon sugar
diterpenes
4 isoprene units ex. vitamin A
How many codons are there?
4 possible bases (A, C, U, or G) and 3 bases to each codon, so: 4³ = 64.
Explain *Hückel's Rule*.
4n + 2 *n* is any integer, but also...it comes from *algebra*, NOT chemistry! The number of π electrons must equal one of the numbers in this series: 2, 6, 10, 14, 18....and so on. For example, we can find aromatic molecules with 2 pi electrons, 6 pi electrons, 10 pi electrons, 14 pi electrons, 18 pi electrons, and so on. But we have never found aromatic molecules with 0, 1, 3, 4, 5, 7, 9, 11, 12, 13, 15, 16, 17 (and so on) pi electrons. Those numbers are not in the series. *To reprise*: the # of pi electrons in an aromatic molecule will always be found in the series [2, 6, 10, 14, 18 ...and so on] When all other requirements of an aromatic compound have been met, you can evaluate the # of π electrons in your molecule and see if that # satisfies this rule. Really, it's just a matter of matching that # to any of the values in this series.
DNA is always read in what direction?
5' ⟶ 3'
The parental strand on the *lagging strand* has what polarity?
5' ⟶ 3' DNA Polymerase can't *read* this (it likes 3' ⟶ 5'), so it creates Okazaki fragments and all the time the replication fork keeps moving forward, so DNA polymerase must back track and fill in the gap ✴︎ See pic: *Light pink* = direction of *lagging parent* strand (flows in the opposite direction than what DNA polymerase can read. Therefore, it must "go back to read" and then synthesize from there, "go back" and synthesize the daughter strand (dark blue) in the 5' ⟶ 3' which is obviously antiparallel *Rainbow* = the direction DNA polymerase *reads*, not synthesizes. Notice that the leading strand's parent strand (*dark red*) has 3' ⟶ 5' polarity, just the way DNA polymerase likes it, so it can just keep moving forward. Again, the rainbow is the direction it *reads* and then synthesizes this daughter strand 5' ⟶ 3' (shown in gold)
In what direction does *primase* add primer to be *paired* with the parent strand.
5' ⟶ 3' Explanation: parent strand is antiparallel, so for the leading strand (bottom), when we look at it from left to right, it is going 3' ⟶ 5', so that the daughter strand across from it from the same perspective (left to right) would be going 5' ⟶ 3', and this is the direction (left to right) *primase* lays down primer ahead of DNA polymerase. for the *lagging strand* (top), its parent strand goes 5' ⟶ 3' from left to right, so the daughter strand across is going 3' ⟶ 5' from left to right. Therefore, *primase* must add primer in the opposite direction (right to left) in order to work because it can only lay down primer from 5' ⟶ 3'. Pic shows the direction *primase* works to lay down primer on both *daughter strands*. It places primer at the 5' because its job is to *start* a fragment, so that DNA polymerase can come and add to the 3' end to finish up.
What is the complementary strand for 5'-ATCG-3'?
5'-CGAT-3' Remember: DNA is always written from 5' to 3' Steps... ① *FLIP* ⟳: Find the complementary sequence: 5'-ATCG-3' ┉┉┉┉┉ 3'-TAGC-5' ^*but this isn't your answer* ② *MIRROR* ↔︎ : Reverse the sequence so that you can properly write the sequence from 5' ⟶ 3': 3'-TAGC-5' ┇ 5'-CGAT-3' (*final answer*)
How is the *5' cap* linked?
5'-to-5' triphosphate linkage https://ibb.co/dS4XG9 Note, this is unusual! (methyl groups donated by SAM)
What comprises a *nucleoside*?
5-carbon sugar (*pentose*) + nitrogenous base ✴︎covalent linkage (*glycosidic bond*) b/w base and C-1' of the sugar
The *Edman degradation* can cleave and sequence proteins of up to ________ amino acids.
50 to 70
How many *distinct* tripeptides can be formed from 1 valine molecule, 1 alanine molecule, and 1 leucine molecule.
6
How many pi electrons are in an imidazole ring?
6 (follows Huckel's 4n + 2 rule) Explanation: 2 from each π bond (total of 4) and the lone pair on the *pyrrole-like nitrogen* (dark blue) ✴︎the pink lone pair of electrons on the *pyrrole-like nitrogen* (dark blue) are delocalized in the ring because of the possible resonance structure ✴︎the light blue lone pair of electron on the *pyridine-like nitrogen* (red) do *not* participate in resonance, because it's the double bond right there that participates; thus the light blue lone pair is *localized* to that N atom (see how they occupy their own orbital in the pic) and the pink double bond can be shunted out of the ring to become a lone pair (so it's participating!) ✴︎Note, the π electrons are in pink in the figure, and shown to delocalize in the ring on the far right.
triterpenes
6 isoprene units can be converted to cholesterol and various steroids
*G Protein-Coupled Receptors (GPCR)* are characterized by their ____ membrane-spanning α-helices.
7
tetraterpenes
8 isoprene units ex: carotenoids group (i.e. beta-carotene and lutein)
Glucose is bound by both glucokinase (Km = 5 mM) and hexokinase (Km = 0.1 mM). After a meal, a diner's blood glucose level rises from 1 mM to 5 mM. At this point, which of the following must be true? A. Hexokinase binds more glucose at all times. B. The two enzymes exhaust the substrate pool. C. Km for the overall reaction is 5 × 10⁻⁴ mM². D. Glucokinase is saturated when glucose levels peak.
A Explanation: Hexokinase has a lower Km ⟶ *greater* affinity. The other answers are not necessarily true.
Enhancers are transcriptional regulatory sequences that function by enhancing the activity of: A. RNA polymerase at a single promoter site. B. RNA polymerase at multiple promoter sites. C. Spliceosomes and lariat formation in the ribosome D. Transcription factors that bind to the promoter but not to RNA polymerase
A Explanation: Specific transcription factors bind to a specific DNA sequence, such as an enhancer, and to RNA polymerase at a single promoter sequence. They enable the RNA polymerase to transcribe the specific gene for that enhancer more efficiently. So the enhancer, once activated by transcription factors, sets a cascade into motion that helps RNA polymerase do its job.
What modifications will be made to a nascent RNA transcript before it exits the nucleus? A. A poly-A tail will be added. B. A 3' guanosine gap will be added. C. Exons will be excised D. Chaperones will assist with folding
A Explanation: The post-transcriptional modifications made to the pre-mRNA are the addition of the poly-A tail and *5'* guanosine cap, as well as the splicing of *introns*. The only answer choice that matches this is (A). (B) is wrong because the guanosine cap is added to the 5' end, not the 3' end. (C) can be eliminated because the process of splicing excises the introns and joins the exons. (D) is also wrong because chaperones are involved in protein folding, which happens *after translation*.
Which ion channels are responsible for maintaining the resting membrane potential? A. Ungated channels B. Voltage-gated channels C. Ligand-gated channels D. No ion channels are involved in maintenance of the resting membrane potential.
A Explanation: The resting membrane potential is displayed by cells that are *not* actively involved in signal transduction. Ungated ("leak") channels permit limited free flow of ions, while the Na+/K+ pump is also active and corrects for this leakage. Voltage-gated and ligand-gated channels *are* involved in cell signaling and in the pacemaker potential of certain cells, but cause *deviation from* - not maintenance of - the resting membrane potential
An *isomerase* is LEAST likely to be co-classified as a(n): A. Ligase B. Hydrolase C. Lyase D. Oxidoreductase
A Explanation: isomerases often involve shifting of electrons and bond-breaking, but they will not ever be classified as ligases.
Ketose sugars may have the ability to act as reducing sugars. Which process explains this? A. Ketose sugars undergo tautomerization. B. The ketone group is oxidized directly. C. Ketose sugars undergo anomerization. D. The ketone group is reduced directly
A Explanation: keto-enol shifts ⟶ converted to *aldose* which can act as reducing sugars.
How is translation different for peptides that will be *secreted*, such as hormones and digestive enzymes?
A *signal sequence* directs the ribosome to move to the ER, so the protein can be translated directly into the lumen of the RER ⟶ Golgi app. ⟶ secreted from a vesicle via *exocytosis* https://ibb.co/gO16B9
sphingosine
A complex *alcohol* backbone for membrane lipids
Triacylglycerols (triglycerides)
A glycerol molecule esterified to three fatty acid molecules (usually, they are all different) the most common form of energy storage in the body Overall nonpolar and hydrophobic Can be seen in oily droplets
calcitriol
A hormone produced from vitamin D that acts in essentially the same manner as parathyroid hormone: ↑ calcium & phosphate uptake in intestines, which promotes bone production
sphingolipid
A lipid containing a sphingosine or sphingoid backbone bound to fatty acid tails; includes... ① ceramide ② sphingomyelins ③ glycosphingolipids ④ gangliosides
micelle
A small particle formed by aggregates of molecules with both polar and nonpolar segments (e.g., soap); the polar ends of these molecules point outward toward a polar solvent (e.g., water) while the non-polar ends will point inward toward a nonpolar solute (dirt/oil/grease). This allows the nonpolar substance to be washed away by the polar substance.
What bases can hypoxanthine (I), which is found in some tRNA, base pair with?
A, C, or U
Name the 5 nucleotides in order of increasing # of phosphate groups (and with both RNA & DNA forms).
AMP/dAMP ⟶ ADP/dADP ⟶ ATP/dATP GMP/dGMP ⟶ GDP/dGDP ⟶ GTP/dGTP CMP/dCMP ⟶ CDP/dCDP ⟶ CTP/dCTP UMP/dUMP ⟶ UDP/dUDP ⟶ UTP/dUTP dTMP ⟶ dTDP ⟶ dTTP
List the 5 nitrogenous bases.
Adenine Guanine Cytosine Uracil Thymine
List the *purine* bases.
Adenine and Guanine Mnemonic: *PUR*e *A*s *G*old and you need *two* gold wedding *rings* for a wedding
List all nucleosides, first with the RNA form, then the DNA form.
Adenosine/Deoxyadenosine Guanosine/Deoxyguanosine Cytidine/Deoxycytidine Uridine/Deoxyuridine (note, *not* found in DNA) Deoxythmidine (there is no "thymidine" because this base is only in DNA).
What is the role of *single stranded binding proteins*?
After *helicase* unpairs the strands, they are *sticky*, so these proteins are required to hold the strands apart Also protect from degradation of DNA by *nucleases*
Why are DNA repair mechanisms mostly active during interphase?
Aimed at *preventing* propagation of error into daughter cells during mitosis.
How do you convert a Fischer projection to a Haworth projection?
All groups on the right of the Fischer projection will go on the bottom of the Haworth, and all groups on the left will go on top. Mnemonic: "down right uplefting"
Enzymes in the same class share what characteristics? A. Reaction mechanism B. General function C. Substrate conformation D. All of the above
B
For the process of transcription the template strand is the: a. Sense strand of DNA b. Antisense strand c. Either strand of DNA
B
Gene regulation in prokaryotes is primarily *negatively regulated*. This means when the *repressor* is bound to the operator: a. Transcription is increased b. Transcription is inhibited c. Transcription is unchanged
B
In an experiment, RNA was extracted from dendritic cells that produce cytokines; however, it was then converted to cDNA before undergoing PCR to quantify RNA levels of the cytokines. Why might this step have been included in the experimental procedure? A. RNA bases can make only double hydrogen bonds with one another, whereas DNA bases can make triple hydrogen bonds. B. DNA is more stable than RNA and can better maintain its integrity during PCR. C. DNA is the molecule in which genetic information is encoded, not RNA. D. cDNA forms more stable complexes with RNA than does genomic DNA.
B Explanation: (A) is false, since RNA can also form triple bonds (b/w C and G). (C) is true but isn't a reason to use cDNA instead of RNA in the way this experiment does. Even though genetic information is stored (in the long-term) by DNA rather than RNA, the particular cDNA from this experiment would hold exactly the same genetic information as the RNA it was made from; there would be no *informational* advantage to converting to cDNA. (D) fails because the question is not asking for a comparison between cDNA and DNA, and they are not structurally different anyway. Also, DNA routinely interacts with RNA during transcription, so their complexes must be stable. (B), however, offers a plausible reason, since the question stem specifically told us the RNA was converted to cDNA prior to going through PCR, it makes sense this was a calculated move to maintain the integrity of the molecules because the cDNA was more stable in the process than RNA would be.
Adding concentrated strong base to a solution containing an enzyme often reduces enzyme activity to zero. In addition to causing protein denaturation, which of the following is another plausible reason for the loss of enzyme activity? A. Enzyme activity, once lost, cannot be recovered. B. The base can cleave peptide residues. C. Adding a base catalyzes protein polymerization. D. Adding a base tends to deprotonate amino acids on the surface of proteins.
B Explanation: Bases *can* catalyze peptide bond hydrolysis, so B is correct. A is incorrect because there are some cases where enzyme activity may be recovered. Finally, D is true, but fails to explain loss of enzyme activity.
Amino acids are dissolved in a basic solution that is gradually titrated with HCl. Which of the following amino acids would require the most added HCl before its positively charged form predominates in solution? A. Lysine B. Aspartate C. Glycine D. Glutamine
B Explanation: This question is asking which amino acid requires the most additional acid to become positively charged. This is another way of asking which amino acid is, itself, the most acidic (since acidic compounds, by definition, don't like to hold on to their protons, it's going to take a lot more HCl in solution to get the acidic amino acids to fully protonate). So think about aspartate (D) and glutamate (E), both of which have negatively-charged side chains at physiological pH. (B) matches. (A): K is one of the three basic amino acids, which have positively charged side chains at physiological pH. (C): G is a nonpolar amino acid with an uncharged side chain at physiological pH (just an H), which means that it is less acidic than aspartate. (D): Q is a polar amino acid with an uncharged side chain at physiological pH, which suggests that it is less acidic than aspartate.
The site where a protein binds essential cofactors is most likely to be characterized by: A. An excess of positive charge B. An excess of negative charge C. Nearly neutral charge D. Hydrophobic side chains
B Explanation: most cofactors are metal cations, so they would be attracted to a largely negative active site
Which of the following techniques can be used for *both* native and denatured proteins? - electrophoresis - chromatography
BOTH can be used for both
Where are ions likely located that are not readily accessible in the cytoplasm or extracellular space?
Bound to a binding protein Ex: calcium & magnesium
Total iron binding capacity (TIBC) is a measure of how much iron can be absorbed by the binding proteins in a patient's blood. Assuming that the concentrations of these binding proteins remain constant, the TIBC in a patient with anemia due to iron deficiency would be: A. low, because the patient is deficient in iron. B. low, because the binding proteins would have a decreased affinity for iron. C. high, because the patient is deficient in iron. D. high, because the binding proteins would have a decreased affinity for iron.
C Explanation: In the case of anemia (low Hb concentration in the blood) due to iron deficiency, the binding proteins in blood would be "starved" of iron, which means TIBC would be *high*: TIBC is raised because iron concentration is low. (D) is incorrect because we don't have any evidence from the question that there's anything wrong with the binding proteins: the proteins would, if anything, have an *increased* affinity of iron to "soak it up."
Why might uracil be excluded from DNA but *not* RNA? A. Uracil is much more difficult to synthesize than thymine. B. Uracil binds adenine too strongly for replication. C. Cytosine degradation results in uracil. D. Uracil is used as a DNA synthesis activator
C Explanation: A common DNA mutation is C ⟶ U in the presence of heat. DNA repair enzymes recognize U and correct this error by excising the base and inserting C. This does not happen appreciably in RNA because RNA only exists transiently in the cell. Were U to be used in DNA under *normal* circumstances, it would be impossible to tell if a base *should* be U or if it is a damaged C.
The four subunits of hemoglobin exhibit cooperative binding. As PO₂ of the blood changes entering the pulmonary veins, the Km of hemoglobin towards this substrate: A. Increases B. Decreases C. Remains unchanged D. Could be higher or lower, depending on the saturation
C Explanation: Km describes the affinity of the *enzyme-substrate complex*, and this value is only affected by inhibitors, *not* by changes in substrate concentration. (It's an inherent value, unique to each enzyme-substrate relationship).
The conversion of ATP to cyclic AMP and inorganic phosphate is most likely catalyzed by which class of enzyme? A. Ligase B. Hydrolase C. Lyase D. Transferase
C Explanation: Lyases breakdown a single molecule into 2 molecules *without* the addition of water or the transfer of electrons. Lyases often *form cyclic compounds or double bonds* in the products to accommodate this. Water was not a reactant (eliminating B), Ligases *make* molecules, and no cofactor was mentioned, so the rest are wrong.
How does the gel for *isoelectric focusing* differ from the gel for traditional electrophoresis? A. Isoelectric focusing uses a gel with much larger pore sizes to allow for complete migration B. Isoelectric focusing uses a gel with added SDS to encourage a uniform negative charge. C. Isoelectric focusing uses a gel with a pH gradient that encourages a variable charge. D. The gel is unchanged in isoelectric focusing; the protein mixture is treated before loading.
C Explanation: When a protein is in a region where pH > pI: it is - and moves toward the anode. When it's where pH < pI: it is + and moves toward the cathode. When pH = pI: migration halted.
A cell has lost its ability to create splieceosomes due to a mutation. What is now true of this cell? A. The cell can no longer do any posttranscriptional processing. B. The cell can no longer do any splicing as part of posttranscriptional processing. C. The cell can now only do some types of splicing during posttranscriptional processing. D. The cell will not be able to create any function proteins.
C Explanation: While spliceosomes are responsible for *most* of the splicing during posttranscriptional processing, there are other (rarer) forms like *self-splicing* and *tRNA splicing*. Therefore, the cell can now only do some types of splicing, but (A), (B), and (D) are all out of scope in different degrees. (D) is the most out of scope because there is not enough evidence to say that the end product will not be functional with just some type of splicing being performed. (A) is also very out of scope because there are certainly other types of posttranscriptional processing, such as the addition of a poly-A tail (B) is out of scope because, as mentioned, self-splicing and tRNA splicing will not be affected and therefore the cell can still do *some* splicing.
If it is known that a certain enzyme does not stabilize the transition state of a reaction, then how might the enzyme lower activation energy for its catalyzing reaction? A. Raising the energy of the bound products B. Raising the temperature of the reaction C. Raising the energy of the bound reactants D. Increasing the equilibrium constant
C Explanation: one of the 5 ways to lower the Ea besides stabilizing the transition state is to destabilize the reactants, perhaps by putting strain on them (↑ the energy)
Why is irreversible inhibition more permanent than "reversible" noncompetitive inhibition.? A. Irreversible inhibition can entirely stop an enzyme's activity, but a noncompetitive inhibitor cannot. B. Adding more substrate reverses noncompetitive inhibition, but does not reverse irreversible inhibition. C. Removing noncompetitive inhibitors reverses their inhibition, but removing irreversible inhibitors does not. D .Adding more enzyme reverses noncompetitive inhibition but does not reverse irreversible inhibition.
C Explanation: other answers are either partially or completely false: - A. is entirely wrong - B would be correct if it said "Adding more substrate reverses *competitive* inhibition" - D. is wrong because synthesizing more enzyme is the *only* way to "reverse" irreversible inhibition.
A man has high blood pressure partially due to a high-salt diet. What effect does the increased salt concentration in his blood have on enzyme activity? A. Enzyme activity is increased. B. Enzyme activity is decreased. C. Enzyme activity is not changed.
C Explanation: salinity has no effect physiologically, only in vitro studies
Consider a reaction catalyzed by enzyme A with a Km value of 5 × 10⁻⁶ M and Vmax of 20 mmol/min. At a concentration of 5 × 10⁻⁴ M substrate, the rate of the reaction will be: A. 10 mmol/min B. 15 mmol/min C. 20 mmol/min D. 30 mmol/min
C Explanation: Can use the M-M equation, or use logic: At a concentration of 5 × 10⁻⁴ M, there is 100 times more substrate than present at half maximal velocity (log scale, so 2 orders of magnitude = 100x). At high values ([S] >>>>> Km), the enzyme is at or near its Vmax, which the question states to be 20 mmol/min.
What two carbons react in D glucose to create the cyclic sugar?
C1 and C5 In the process, a new OH group (original aldehyde O picks up a proton, creating a single bond b/w them O-H and the OH on C5 is what joins the ring togethe) forms on the anomeric carbon And the carbonyl carbon has also become chiral in the process
Where is the anomeric carbon in *fructose*?
C2
D-mannose
C2 epimer of glucose aldohexose
D-galactose
C4 epimer of glucose aldohexose
Each tRNA has a _ _ _ nucleotide sequence where the AA binds.
CCA
What happens during *cytosine deamination*?
Cellular insult, usually thermal energy, causes *loss of an amino group* from cytosine ⟶ uracil. During DNA replication, U will pair with A rather than G (as it would if the base was still C), so an uncorrected cytosine-to-uracil change can lead to a mutation.
When there is a large quantity of proteins to be separated, which technique is better suited - electrophoresis or chromatography?
Chromatography Explanation: gel can only handle a small volume of protein
Why does *cDNA* lack noncoding regions?
Constructed by reverse-transcribing mRNA, and we know that when DNA is converted to mRNA, only the coding regions (exons) are copied, *not* in the noncoding region (such as introns)
What is the empirical formula for monomeric sugars (monosaccharides)?
C𝘯(H₂O)𝘯
Which of the following is a diastereomer of α-L-threose? I. α-D-threose II. β-L-threose III. β-D-threose A. I only B. II only C. I and II only D. II and III only
D Explanation: By definition, L- and D-sugars have inverted stereochemistry at all chiral centers, and are therefore enantiomers of each other. Therefore, (I) corresponds to α-L-threose's one and only enantiomer, α-D-threose, and since by definition there is only one enantiomer, the other two Roman numerals are diastereomers. To prove these are diastereomers: (II): β-L-threose should have the same stereochemistry as α-L-threose at all chiral centers except for the anomeric carbon (which designates the sugar as α or β). β-L-threose is therefore an anomer of α-L-threose. Anomers are a subtype of epimers, which are diastereomers that differ at exactly one chiral carbon. (III): β-D-threose is the anomer of α-D-threose. This means that β-D-threose is a diastereomer of α-D-threose. Therefore, it can't be an enantiomer to α-L-threose since we already found its one and only enantiomer (I). Specifically, it differs from α-D-threose at the anomeric carbon (again)
The ATP synthase found in mitochondria is a(n): A. Ligase because it involves the formation of a new bond in a nucleotide. B. Transferase because it moves a phosphate group onto ADP. C. Oxidoreductase because ATP is an energy storage molecule. D. Lyase because it involves two small, different molecules
D Explanation: "Synthase" may refer to either ligases or lyases; however, here, ATP is synthesized by using ADP + inorganic phosphate, which are 2 small, different molecules.
A certain cooperative enzyme has four subunits, two of which are bound to substrate. Which of the following statements can be made? A. The affinity of the enzyme for the substrate has just increased. B. The affinity of the enzyme for the substrate has just decreased. C. The affinity of the enzyme for the substrate is at the average for this enzyme class. D. The affinity of the enzyme for the substrate is greater than with one substrate bound.
D Explanation: A is attractive, but here's why A (and B) are wrong: We cannot determine whether the most recent reaction was binding or dissociation, so there's not enough info to infer either way for sure. C is not necessarily true because the increase in affinity is not linear (in other words, it could increase or decrease by logarithmic amounts, etc.) D is the only answer that is *necessarily* true.
Which of the following is NOT involved in cell migration? A. Dynein B. Flagella C. Actin D. Centrioles
D Explanation: Centrioles are composed of microtubules, but are involved in mitosis, *not* cell motility.
When comparing two Lineweaver-Burk plots, the enzyme that produces the graph with the steepest slope will have: A. The fastest reaction rate B. The smallest Km value C. The highest substrate concentration D. The largest Km value
D Explanation: the slope will change when *Km* changes (moves closer to origin) but *Vmax* does not. When does this happen? Competitive inhibition. A competitive inhibitor will *increase* the Km (it takes more substrate to *outcompete* the inhibitor) but will have no effect on Vmax, resulting in a *steeper* slope.
*Southern blot* detects ___________.
DNA Mnemonic: think *south* ￫ the very beginning (DNA)
How can DNA cloning be used to produce *DNA libraries*?
DNA fragments are cloned into vectors and can be utilized for further study
Which prokaryotic DNA polymerase has both *5' ⟶ 3'* and *3' ⟶ 5'* *exonuclease* activity?
DNA polymerase *I* Its jobs ① *5' ⟶ 3' polymerase*: replaces RNA primer *5' ⟶ 3'* with nucleotides on the daughter strand all in one pass *Note*, it does this on both leading and lagging strands, though the picture only shows the lagging. ② *5' ⟶ 3' exonuclease* activity for *removing RNA primers* & *nick repair* (Note, DNA polymerase III lacks this, but it can proofread, see below) ③ *3' ⟶ 5' (reverse) exonuclease*: for *proofreading*
Which polymerase/s is/are the principle replication enzyme, synthesizing the daughter strands in *prokaryotes*?
DNA polymerase III Its jobs: ① *5' ⟶ 3' polymerase*: synthesizes DNA (remember, it requires a template, so it needs the help of *primase* which will lay down RNA primer) ② *3' ⟶ 5' exonuclease*: Proofreading ❗️does *not* have 5' ⟶ 3' exonuclease activity.
Which polymerase/s is/are the principle replication enzyme/s, synthesizing the daughter strands in *eukaryotes*?
DNA polymerases α, δ, and ε Their jobs: • *DNA polymerases α* ￫ ① *5' ⟶ 3' polymerase*: synthesizes DNA by working with *primase* and is involved in the *initiation* of DNA replication at the origins of replication. Once *primase* has created the RNA primer, *Pol α* starts replication, elongating the primer with ~20 nucleotides. ❗️lacks *exonuclease* activity, and has low processivity ◦ *DNA polymerase δ* ￫ ① *5' ⟶ 3' polymerase*: also involved in synthesis on both leading & lagging strands, but its role on the *lagging strand* is important as it functions to fill in gaps left behind when RNA primers are removed with new DNA nucleotides. ② *3' ⟶ 5' exonuclease*: proofreading ❕has high processivity ◆ *DNA polymerase ε* ￫ ① *5' ⟶ 3' polymerase*: also involved in synthesis on both leading & lagging strands, but esp. on the *leading strand*. It is deeply involved in DNA repair via *nucleotide* and *base excision repair* ② *3' ⟶ 5' exonuclease*: some say it also has ability to proofread ❕has high processivity
What is *recombinant DNA*?
DNA produced by combining genetic material from multiple sources in a lab Ex: recombinant human insulin
What enzyme helps counteract the strain induced by positive supercoiling that occurs during the unzipping of DNA by *helicase*?
DNA topoisomerases
What is a *nucleosome*?
DNA wrapped around 8 histone proteins (2 copies of each H2A, H2B, H3, & H4)
How do prostaglandins protect stomach from acid?
Decrease acid production, stimulate cells to secrete bicarb and mucus, stimulate blood flow ❗️That's why NSAIDs can cause ulcers! They function to *block* prostaglandins
Some eukaryotic proteins contain *signal sequences* , which designate a particular destination for the protein. Where will the ribosome move after encountering this sequence?
ER, nucleus, lysosomes, or cell membrane. https://ibb.co/gO16B9
Why is DNA replication called semi-conservative?
Each daughter DNA molecule is composed of one parental strand and one new strand
What is an *anticodon*?
Each tRNA contains a set of three nucleotides called an *anticodon*. It can bind to one or a few specific mRNA codons.
Distinguish between *epimer* and *anomer*.
Epimers are a subtype of diastereomers that differ at exactly one chiral carbon Anomers are a subtype of epimers that differ at the anomeric carbon
Why are *epimers* classified as diastereomers and not enantiomers?
Epimers differ at exactly one chiral carbon, so by definition However, they cannot be enantiomers because they are *not* mirror images of each other. (which would *have* to be inverted at *all* chiral centers) This is the definition of *diastereomer*, so epimer is a subclass.
Why are *cDNA* libraries the most reliable when it comes to... - sequencing specific genes - identifying disease-causing mutations - producing recombinant proteins, or - producing transgenic animals ...?
Even though genomic libraries contain the entire genome of an organism, genes may by chance be split into multiple vectors so the *cloned genes are not necessarily complete sequences*. *basically, it is more focused and a whole lot smaller, so isolating specific *expressed* genes is easier and less prone to error*
True or False: A ketone group can be further oxidized.
FALSE
True or False: H1 is part of a nucleosome.
FALSE Explanation: H1 is the linker, and H1 + nucleosome + linker DNA gets its own term ￫ *chromatosome*
True or False: DNA can be synthesized de novo.
FALSE Explanation: it needs another molecule to "hook on to", which is why *primase* must lay down an *RNA* primer to be directly paired with the parent strand.
True or False: *Spliceosomes* are complexes that facilitate all forms of splicing.
FALSE Explanation: most, but *not* all. Other (rarer) forms: ① self-splicing ￫ introns that form ribosomes and perform the functions of a spliceosome alone ② tRNA splicing
True or False: Km is half Vmax
FALSE Explanation: the *concentration of substrate* that causes the reaction to run at 1/2 Vmax it's *not* a measure of velocity/rate
True or False: Km and Vmax do not apply to ion channels, only enzymes.
FALSE Explanation: these parameters are also applicable to transporters such as these.
True or False: *Peptidyltransferase* is a protein
FALSE It is actually the rRNA of the large ribosomal subunit ✴︎ remember, we said the ribosome had *catalytic* activity and this is it!
True or False: Prokaryotic DNA contains nucleosomes.
FALSE No histones, therefore cannot form nucleosomes
True or False: a transcription factor must bind near the promoter in order to affect transcription.
FALSE binding sites for a TF can be very far away and *still affect transcription* *The flexibility of DNA is what allows transcription factors at distant binding sites to do their job* The DNA loops like cooked spaghetti to bring far-off binding sites and transcription factors close to general transcription factors or "mediator" proteins. In the cartoon attached, an activating transcription factor bound at a far-away site helps RNA polymerase bind to the promoter and start transcribing
True or False: All sphingolipids are phospholipids.
FALSE they can have other types of linkages, like glycosidic bonds, so only some are phospholipids
True or False: All genes that are transcribed are translated.
FALSEEEEE Genes that do not code proteins also are transcribed (translation means end product is protein!): • rRNA, ribosomal RNA ￫ Catalyze protein synthesis by facilitating the binding of tRNA (and their amino acids) to mRNA. • tRNA, transfer RNA ￫ Transport amino acids to mRNA for translation. • snRNA, small nuclear RNA ￫ Combine with proteins to form complexes used in RNA processing (spliceosomes used for intron removal).
True or False: Every carbon in a monosaccharide carries a hydroxyl group
False Every carbon *other than the carbonyl* carbon will carry a hydroxyl group in a straight chain monosaccharide and then every carbon *except the chiral carbon with the highest number* in a cyclic monosaccharide
True or False: Steroid hormones affect gene transcription by binding directly to DNA
False Explanation: Steroid hormones are produced in endocrine glands & travel in the bloodstream to bind to high-affinity receptors in the nucleus. The hormone's *receptor* binds to *DNA*, but the hormone itself does not
True or False: *Km* can be changed by altering the concentration of substrate or enzyme
False Explanation: it's an inherent property of the enzyme-substrate system
True or False: DNA ligase has proofreading ability.
False Explanation: this is why the lagging strand is more prone to mutation, because ligase is more active on this strand, closing the gaps b/w Okazaki fragments and no way to proofread.
True or False: *Lyases* only cleave molecules.
False! Explanation: Remember, enzymes speed up both the forward and reverse reactions, so they can also put molecules together
What happens to the α-subunit when the GPCR is activated?
First, GDP is replaced with *GTP* Then, α-subunit can dissociate from the β and ɣ subunits
How do you convert a Fischer projection to a chair conformation?
Fischer ⟶ Haworth ⟶ Chair
What is *wobble*?
For AA with multiple codons, the first two bases are usually the same, and the *third base in the codon is variable*. The pairing b/w the 3' base of the codon and the 5' base of the anticodon does not follow Watson & Crick's strict rule of base pairing. Instead, we see strange pairing like G-U, G-A, C-U. Even I (hypoxanthine) can be found, and it pairs with A, C, or U (just not G). Wobble is an evolutionary development designed to protect against mutations in the coding regions of DNA.
How is *cDNA* formed?
Formed from a processed mRNA strand by *reverse transcription*. (*c* = *c*omplementary)
In what direction will charge flow during *electrophoresis* of DNA?
From *cathode* (-) ⟶ *anode* (+) Explanation: cations migrate towards the cathode (-) & anions migrate towards the anode (+) DNA is negatively-charged (phosphate backbone) so it will be attracted to the + anode
In what order is a peptide synthesized by ribosomes during translation?
From the N-terminus to the C-terminus (left to right) ✴︎by convention, peptides are read the same way, from N to C
What is the usual cleavage site on signal sequences?
GLY or ALA residues ⟶ small side chains
Glycerophospholipids (phosphoglycerides)
Glycerol backbone bound by ester linkages to *two* fatty acids and phosphodiester link to a highly polar head
*Base excision repair* occurs during what phase of the cell cycle?
G₁ and G₂
*Nucleotide excision repair* occurs during what phase of the cell cycle?
G₁ and G₂
Mismatch repair occurs during what phase of the cell cycle?
G₂
What acts to link *nucleosomes*
H1 it seals off the DNA as it enters and leaves the nucleosome, adding stability to the structure
During a transition from the *R* state ⟶ *T* state, what happens to the substrate?
Having been bound, now the substrate dissociates from the remaining subunits. This transition occurs when *concentration of substrate is low*, meaning that the substrate (e.g. oxygen) is needed in the tissues, *not* on the transport protein, so it "jumps off"
Which of the following receptors utilize second messenger systems? I. Ligand-gated ion channels II. Enzyme-linked receptors III. G protein-coupled receptors
II and III only
Which has higher processivity - DNA polymerase I or III?
III Explanation: DNA Pol III is the enzyme primarily responsible for DNA replication and forms a replication complex with extremely high processivity. The related DNA polymerase I has 2 forms of exonuclease activity on top of its *5' ⟶ 3'* polymerase activity, which involves replacing RNA primers w/ DNA. Thus, its primary function in is to create many short DNA regions rather than a few very long regions. https://en.wikipedia.org/wiki/Processivity
When a protein is said to be *monomeric*, what can be assumed about its quaternary structure?
IT DOESN'T HAVE ANY
Ligases may go by either the name "synthase" or synthetase". Which one *necessitates* that it is a ligase?
If it's -synthetase, it *must* be a ligase If it's -synthase, it could also be a lyase, so it's not a dead giveaway
How does D glucose convert into a cyclic sugar?
In some cases, OH and carbonyl groups on the same molecule are able to react with one another in an intramolecular reaction Shown is the α anomer, because the OH is down. When a straight-chain monosaccharide forms a cyclic structure, the carbonyl oxygen atom may be pushed either up or down, giving rise to two stereoisomers
What type (D or L) of amino acid is cysteine and why?
It is still *L*; however, it is different from all the other chiral AA's because it has the absolute configuration (R), *not* (S). This is because the -CH₂SH group has priority over the -COOH group.
What is usually required in order for a ligand that is present in low quantities to have a strong action?
It should initiate a *second messenger cascade system*
What happens to the Lineweaver-Burk plot during *mixed inhibition*?
It's a mix of competitive and uncompetitive: The *apparent Vmax* decreases, because the inhibitor is capable of preventing catalysis *regardless*: If it prefers the enzyme ￫ A mixed inhibitor can bind to the enzyme at an allosteric site either before or after the substrate binds. Therefore, it CANNOT be overcome by increasing the substrate concentration because it will still be able to inhibit the enzyme. If it prefers the E-S complex, Vmax will decrease as less enzyme is available because it and the substrate are essentially "locked together". The change in *Km* will vary: If the inhibitor binds preferentially to the *enzyme*, then apparent Km will increase If it likes the *E-S complex* more, then apparent Km decreases *Therefore, the intersection of these plots will not lie on either axis*.
What are the most prevalent extracellular proteins?
Keratin, elastin, collagen
What is the slope of the line on a *Lineweaver Burk* plot?
Km/Vmax *May not be obvious, but i proved it through a very long calculation so just memorize it!
What are the only amino acids that are *not* encoded by multiple codons?
Methionine ⟶ always AUG Tryptophan ⟶ always UGG
NANA
N-acetylneuraminic acid; a/k/a sialic acid component of the polar head groups of *gangliosides*
The *Edman degradation* selectively and sequentially removes the ____-terminal amino acid of the protein, which can be analyzed via mass spectroscopy.
N-terminal
Does RNA polymerase need a primer?
NO
Is the resonance in the peptide bond adequate for UV absorption?
NO
Do *transferases* eliminate any substrate in a reaction?
NO Explanation: While transferases might change what the initial compound *looks like*, both initial compounds are technically still present after the transferase reaction
After processing of mRNA, it is ready to be transported into the cytoplasm for *protein translation*. Will the entire mRNA molecule be translated?
No, there are still some regions that won't be translated called (*UTRs*). They exist at the 5' and 3' edges of the transcript, because the ribosome initiates translation at the start codon (always *AUG*) and will end at a stop codon (*UAA*, *UGA*, or *UAG*)
Does RNA polymerase proofread?
No, unlike DNA polymerase
What is the equation for the pI of a basic amino acid?
Note, the pI for such an AA will be higher than other types (well above 6)
What is the equation for the pI of an acidic amino acid?
Note, the pI for such an AA will be lower than other types (well below 6)
In a repressible operon, if the metabolite is present, transcription is ________.
OFF Explanation: if your body has enough of it, it won't need to make so much of it. The metabolite is the corepressor that will bind to the repressor and allow it to bind to the operon and block DNA pol
In an inducible operon, if the metabolite is present, transcription is ________.
ON Explanation: it *induces* the gene to be turned on by binding the repressor and freeing the operon for DNA pol to bind
What is the role of *DNA ligase*?
Once DNA polymerase is done synthesizing the *Okazaki fragments* on the *lagging strand*, *DNA ligase* comes & uses the energy from hydrolyzing ATP, the fragments are fused together to create a new daughter strand.
In *affinity chromatography*, beads in the column are coating with receptors or antibodies that bind a specific protein, thus *retaining* it in the column. How, then, can this "stuck" protein be collected?
Option ①: elute it by washing the column with a free receptor/target/antibody, which will *compete* with the bead-bound receptor and ultimately free the protein from the column Option ②: create an eluent with a specific pH or salinity level that *disrupts the bonds* b/w ligand and desired protein.
When is *hybridization* used in DNA technology?
PCR and Southern Blotting
What is *ubiquitination*? How is it used to regulate the translation of proteins?
Proteins can be tagged for degradation by the addition of a chemical marker called ubiquitin. Ubiquitin-tagged proteins are taken to the *proteasome*, or "recycling center" of the cell, and broken down into their component parts. Ubiquitination is an important way of controlling the persistence of a protein in the cell
What is a *conjugated* protein and what are their purpose?
Proteins with covalently-attached *prosthetic groups* (nucleic acid, lipid, or carbohydrate "tags") added to them that indicate that these proteins should be *directed to the cell membrane* (esp. lipid tags) *or to specific organelles* (ex: the lysosome). Also, adding a *cofactor may help provide the activity of the protein* (ex: heme group in Hb needed to bind O₂).
How is the eukaryotic rRNA synthesized?
RNA polymerase I transcribes the 28S, 18S, and 5.8S rRNAs as a single unit within the *nucleolus* ⟶ 45S *pre-rRNA* This precursor is processed to become the 18S rRNA of the 40S small ribosomal subunit *and* the 28S and 5.8S rRNAs of the 60S large ribosomal subunit. RNA polymerase III transcribes the 5S rRNA, which is also found in the 60S large ribosomal subunit, occurring *outside the nucleolus* Result: a 60S subunit and a 40S subunit which won't join until protein synthesis to form the whole 80S ribosome (They will dissociate and be reused after they perform their role in translation)
Which eukaryotic enzyme removes RNA primers via *exonuclease* activity?
RNase H Its job: removes RNA primer *5' ⟶ 3'* on the daughter strand and replaces it with nucleotides all in one pass ✴︎ Pic shows *RNase H* (rainbow) *removing* primer *5' ⟶ 3'*, and then *DNA polymerase δ* (blue/green) goes back and fills in gaps! ✴︎✴︎In contrast, in prokaryotes *DNA Polymerase I* does both the removing and the filling in of gaps
Proofreading by DNA polymerase occurs during what phase of the cell cycle?
S prokaryotes: DNA polymerase I, II, and III eukaryotes: DNA polymerase δ and possibly ε
Show how *DNA polymerase I* uses its *5' ⟶ 3' exonuclease* activity.
See pic *Note*, this is different from it's has *3' ⟶ 5'* exonuclease activity (proofreading)
What feature of prokaryotic mRNA serves an equivalent function of the 5' cap seen in eukaryotes? Why is it more suitable to prokayotes?
Shine-Dalgarno sequence Bacterial genes are often transcribed in operons so one bacterial mRNA can contain the coding sequences for several genes. A Shine-Dalgarno sequence *marks the start of each coding sequence*, letting the ribosome find the right start codon for each gene
How can the Michaelis-Menton equation be simplified when the system is at very low substrate concentrations? State the answer in terms of *kcat*.
Since Vmax = kcat[E] Plug kcat[E] in the equation Then [S] in the denominator can be assumed to be insignificant, so it is essentially like adding Km + 0 So the denominator simplifies to Km
What are *siRNA*s?
Small interfering RNA, sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules, 20-25 base pairs in length, similar to miRNA, that operate within the RNA interference pathway
Downstream effects of prostaglandins
Smooth muscle function influence on sleep/wake cycle elevation of body temp during fever and pain
Glycolipids (glycosphingolipids)
Sphingolipids with head groups composed of sugars bonded by glycosidic linkages ￫ found on outer surface of plasma membrane ￫ 3 types (first two are *neutral* at phys. pH) ① *cerebrosides* - neutral ② *globosides* - neutral ③ *gangliosides* - negative ❗️*NOT* phospholipids
Why is the thiol group in cysteine prone to oxidation?
Sulfur is larger and less electronegative than oxygen, so the S-H bond is weaker than the O-H bond
Write the complementary strand for GATTACA.
TGTAATC Explanation: No hints given on orientation, so it's assumed that you know we are given 5' ⟶ 3 So first find the complement (*flip* ⟳): GATTACA ┉┉┉┉┉ CTAATGT Then format it in the 5' ⟶ 3 (*mirror* ↔︎): CTAATGT ┇ TGTAATC
Which nucleotide in a gene is numbered 0?
TRICK QUESTION The first base transcribed from DNA to RNA is +1 and any bases to the *left* (upstream, towards 5') are given negative numbers -1, -2, -3, etc. and bases to the *right* are given positive numbers +2, +3, +4, etc.
What RNA polymerases are involved in prokaryotic transcription?
TRICK QUESTION There's only one that transcribes everything (mRNA, tRNA, and rRNA) and it's just called *RNA polymerase*
Which stage of protein synthesis does *not* require energy?
TRICK QUESTION All stages require large amounts of energy
What is the *telomere* sequence?
TTAGGG
Which polymerase is used in the *polymerase chain reaction*?
Taq polymerase Explanation: The thermal cycling that regulates this reaction would denature human enzymes, so this unique version from the Thermus aquaticus is used instead because it can withstand the extreme temperatures.
Distinguish between *mutarotation* and *anomerization*.
The difference is really subtle. *Anomerization* (or anomerism) refers to the formation of an anomer from the straight-chain form of the sugar. *Mutarotation* is the interconversion between the α-anomer and the β-anomer
Histidine is classified as a amino acid with a *positively-charged (basic)* side chain, though in the body it is often neutral overall. How does histidine acquire a positive charge?
The pKa of the side chain is relatively close to 7.4 (~6), so at physiologic pH, 1 N atom is protonated and the other isn't (*overall uncharged*) The second N atom can become protonated *under more acidic conditions*, giving the side chain a + charge. (see pic)
Show how carbohydrates can undergo *esterification*.
The sugar has the -OH and here it's reacted with acetic anhydride (carboxylic acid derivative) to yield an ester
How do transport kinetics differ from enzyme kinetics?
They are *similar* in that *transport kinetics* also display both Km and Vmax values and can be cooperative They *differ* in that transporters do *not* have analogous Keq values for reactions *because there is no catalysis*
What are *transcription factors*?
They are molecule complexes that control which genes are turned on and off. They help ensure that the right genes are expressed in the right cells of the body, at the right time Transcription factors that are *activators* boost a gene's transcription, while *repressors* decrease transcription They search for transcription factor binding sites, which can be *promoter* and *enhancer* in the case of activators or *silencers* in the case of repressors. Note, the 💭in the image is just an example of "logic"/*combinatorial regulation* exhibited by certain genes. It varies based on the gene.
What is a mnemonic for remembering the structures for *leucine* and *isoleucine*?
They have the same side chain length, the only difference is in the placement of the methyl group: *leucine* has the methyl on the 2nd carbon on the side chain, pointing down ("*l*ook down") *isoleucine* has the methyl on the 1st side chain carbon, pointing *up* ("*e*yes up") ✴︎in fact, they are *constitutional isomers* of each other!
What happens if you add additional base to an amino acid solution that has already been fully deprotonated?
This will only increase the pH further
True or False: *single-stranded* DNA-binding proteins are present, and serve the same function, in both prokaryotes and eukaryotes.
True
True or False: Detergents can denature a protein.
True
True or False: Heterochromatin is composed of highly repetitive DNA sequences.
True
True or False: Motor proteins also display enzymatic activity
True Ex: ATPases
True or False: On a *Lineweaver Burke plot*, as you stray from the x-axis in either direction, Km decreases.
True Explanation: in either direction, it is moving away from [S] = infinite, so it is *decreasing*
True or False: With cooperative binding, each subsequent substrate that binds will do so with a higher affinity than the previous one.
True Explanation: This is the definition of cooperativity
True or False: *Ligases* are the only class of enzymes that absolutely require ATP.
True Explanation: Though others may use ATP in their mechanisms, ligases are the only ones that require it. Mnemonic: L*ig*ases = *big* molecules; ATP needed
True or False: For both DNA and protein electrophoresis, the longer/bigger the strand, the slower it migrates through the gel
True Explanation: in either case, smaller fragments are less impeded by the gel matrix and move faster through the gel
True or False: Prokaryotic DNA lacks telomeres
True Explanation: only eukaryotic DNA has telomeres
True or False: All glycerophospholipids are phospholipids
True they have a glycerol backbone joined to fatty acid tails by ester linkages and a head group joined by *phosphodiester linkages*
True or False: Hypertonic concentrations can denature a protein.
True ✴︎*hypo*tonic concentrations will *not* have this effect, though.
True or False: Enzymes increase the rate of both the forward and reverse reactions.
True! *Notice how hydrolases and lyases can join or split molecules!
What are the primary cytoskeletal proteins?
Tubulin and actin
What is important to realize about the *amide* side chains in asparagine and glutamine?
Unlike the amino group common to all AAs, the amide nitrogens do *not* gain or lose protons with changes in pH **they do not become charged
On what types of cells are *selectins* expressed?
WBCs & endothelial cells Explanation: `big role in host defense (i.e. inflammation & WBC migration)
Show how DNA polymerase *proofreads*.
When an incorrect base pair is recognized, DNA polymerase *reverses its direction* by one base pair of DNA and excises the mismatched base
When do *aldonic acids* form?
When monosaccharides are switching b/w anomers, they spend a brief time in the open chain aldehyde form. At this point, they can be *oxidized* to carboxylic acids, which in this case, they are termed *aldonic acids*
What is the most common (75%) and reliable method of determining protein *structure* after isolation?
X-ray crystallography other option is NMR (25%)
Can *ketoses* be reducing sugars?
Yes, all monosaccharides can be, but ketoses *must first* tautomerize to the *aldose* form
What can be assumed about the concentration of enzyme when dealing with questions about *saturation kinetics* on Test Day?
[enzyme] is constant, it's the [substrate] that changes
How is *reaction velocity* (y-axis) measured?
[product]/time
What effect do *missense (truncation) mutations* have on protein synthesis?
a codon is produced that codes for a different amino acid. The effect varies depending on the new codon, but the primary sequence is again affected
What is an *enol*?
a compound with a double bond and an alcohol group
As new phosphodiester bonds are made during DNA replication, what molecule is released?
a free *pyrophosphate* (*PPᵢ*) *Note*, RNA synthesis occurs by the same mechanism!
What is a *glycoside*?
a glycoside is an acetal or ketal formed from the reaction of cyclic monosaccharides with alcohols in the presence of acid a *carbohydrate* (glycone) + noncarbohydrate (aglycone)
liposome
a minute spherical sac of phospholipid molecules enclosing a water droplet, especially as formed artificially to carry drugs or other substances into the tissues. *transport*
Define *oncogene*.
a mutated gene that causes cancer
What effect do *nonsense (truncation) mutations* have on protein synthesis?
a premature *stop codon* in produce, in effect shortening the primary AA sequence of the protein
What is a *cistron*?
a region of DNA that encodes a single polypeptide chain
lye
a strong basic solution of sodium or potassium hydroxide
What do all phospholipids have in common?
a tail composed of long-chain fatty acids they differ, though, in saturation and length
What are *microRNA*s (miRNAs)?
a type of *small regulator RNA* that can cause an mRNA to be chopped up or block translation
α-D-glucose
a/k/a α-D-glucopyranose
β-D-glucose
a/k/a β-D-glucopyranose
What do *partition coefficients* measure?
ability of a molecule to dissolve in a polar vs. nonpolar environment. in essence, a measure of *lipophilicity*
What biological purpose do micelles serve?
absorb fat-soluble vitamins and complicated lipids (i.e. lecithin) by increasing the surface area available for lipolytic enzymes
Most *nucleoproteins* are (acid/base) soluble.
acid
What is the role of ATP in kinase-catalyzed reactions?
act as a *coenzyme donor* giving up a phosphate group
What is the most abundant protein in eukaryotic cells?
actin
___________ is a protein that makes up microfilaments and the thin filaments in myofibrils.
actin
Oncogenes are most likely to result in cancer through __________.
activation
When does *splicing* occur?
actually can occur co-transcriptionally or shortly after so it's partly *posttranscriptional* and partly *co-transcriptional*, depending on the context.
Besides condensation/dehydration, peptide bond formation is also classified as...
acyl substitution ✴︎Note, this is the complex version of the mechanism. Simplified, the nucleophilic *amino group* on one AA attacks the electrophilic *carbonyl group* on another AA, knocking off the -OH and forming the amide peptide bond
How could one test specifically for glucose when using one of the reagents that test for general reducing sugars?
add in *glucose oxidase* w/ either the Tollens' or Benedict's reagent, because this enzyme does not react with other reducing sugars
According to the *Lock and Key Model* of enzyme action, any enzymatic change must occur (before/after) binding substrate.
after
When is the *poly(A)-tail* added?
after transcription specifically, right after the mysterious termination signal where an enzyme cleaves the primary transcript 10-20 nucleotides downstream
What is the preferred gel for *DNA* electrophoresis?
agarose
What is a mnemonic for remembering the structures for *alanine* and *valine*?
alanine starts with an -A and is pretty simple: just a methyl attached to the amino *v*-aline is just alanine with a *v* added to it.
How does *Benedict's reagent* test for reducing sugars?
aldehyde is oxidized, forming Cu₂O (a red solid)
When the aldehyde group of an aldose is reduced to an alcohol, the compound is considered an ___________.
alditol ex: eryhritol 😄
D-glucose
aldohexose
Glucose is what type of sugar?
aldohexose
What type of pH is used to denature DNA?
alkaline Explanation: acid would break the phosphodiester bonds too, and we just want to separate the double helix.
Which polymerases in prokaryotes have the ability to proofread?
all 3 (I, II, and III)
What is a *replisome*?
all components of DNA replication at a replication fork *the whole picture*
What is the relationship between phospholipids and glycerophospholipids?
all glycerophospholipids are phospholipids but *not* all phospholipids are glycerophospholipids
Decode this DNA sequence: pApTpG
all it is is showing where the phosphate groups are (see figure)
What would be the effect of an *activator* on an allosteric enzyme?
allosteric activators favor the R state, so all subunits will be in the R state
What would be the effect of an *inhibitor* on an allosteric enzyme?
allosteric inhibitors favor the T state, so all subunits will be in the T state
How is *wobble* beneficial evolutionary-speaking?
allows for a single tRNA (with only one anticodon) to pair with four codons for the same amino acid. If each of the 61 codons for AA required a distinct tRNA, cells would contain 61 tRNAs. Wobble fixes this so that fewer tRNAs are required to translate the genetic code.
What allows eukaryotes to encode more different proteins than we have genes in our DNA (a/k/a allows for *variability*)?
alternative splicing Explanation: In the diagram attached, the same pre-mRNA can be spliced in three different ways, depending on which exons are kept. This results in three different mature mRNAs, each of which translates into a protein with a different structure
What is the relationship b/w *kcat* and *Vmax*
always directly proportional
In an inducible operon, if the operator is mutated, what happens to transcription of the gene?
always on Explanation: repressor can't bind if the operator is mutated
Peptide bonds are classified as ___________ bonds.
amide
A tRNA molecule bound to an amino acid is called a ___________.
amino-acyl tRNA
What is the first error-checking step in translation?
aminoacyl-tRNA synthetases ⟶ they check their work, and if the incorrect AA has been linked to a particular tRNA (remember, each is specific for each of the 20 AAs), the enzyme will remove the AA from the tRNA, and try again using the correct amino acid
cholesterol
amphipathic steroid that helps mediate membrane fluidity by interacting with both the heads and tails
Plants predominantly store starch as...
amylose
Biologically, which forms of the acidic amino acids (and other biomolecules) will predominate?
anion form (*-ate*) *not* the acidic (*-ic acid*) form Explanation: most acids in cells exist in the deprotonated form because the pH is closer to neutral
In *gel electrophoresis* of *DNA*, the molecules all migrate towards the __________.
anode (+) Explanation: DNA has a - charge.
Amylopectin (starch)
anomeric configuration: α-D-glucose molecules glycosidic bond type: both α-1,4 *and* α-1,6
Glycogen
anomeric configuration: α-D-glucose molecules glycosidic bond type: both α-1,4 but *more* α-1,6 than amylopectin ⟶ highly branched
Amylose (starch)
anomeric configuration: α-D-glucose molecules glycosidic bond type: α-1,4
Cellulose
anomeric configuration: β-D-glucose molecules glycosidic bond type: β-1,4 other interactions: hydrogen bonds hold chains together for support
___________ refers to ring closure of a monosaccharide, creating an anomeric carbon.
anomerization
What are the most prominent type of proteins found in the immune system?
antibodies (*immunoglobulins*)
Why do *noncompetitive inhibitors* have no effect on Km?
any copies of the enzyme that are still active maintain the same affinity for their substrate! (these inhibitors bind at an allosteric site *and* they do not lock in the E-S complex like uncompetitive inhibitors do) Only Vmax decreases because less enzyme is available overall to facilitate a faster reaction rate.
What is an enzyme called if it is missing necessary cofactors and coenzymes?
apoenzyme
*Trypsin* cleaves at the carboxyl end of which amino acids?
arginine and lysine
Purines and pyrimidines can be classified as biological...
aromatic *hetero*cycles
What makes nucleic acids exceptionally stable?
aromaticity
vitamin D (cholecalciferol)
as calcitriol ￫increases calcium & phosphate uptake in intestines (= bone production)
When is a molecule especially sensitive to pH changes?
at the isoelectric point (when it's neutral) *the titration curve is nearly vertical
Where are *transgenic mice* altered?
at their *germ line* by introducing a cloned gene (called a *transgene*) into fertilized ova or into embryonic *stem cells*
What are *restriction enzymes*?
bacterial enzymes that cut DNA at specific palindrome sequences, sometimes producing offset cuts ("sticky ends"). ✴︎the same restriction enzyme that isolated the sequence may also be used to cut the vector so that the fragment can be inserted directly into it.
Why is the genetic code described as *degenerate*? How is it unambiguous?
because AAs can be specified by more than one codon iow, an amino acid may have more than one codon Ex: Alanine ⟶ GCU, GCC, GCA, GCG However, it's unambiguous because *each codon specifies only one amino acid* Ex: If we see GCU, it can *only* be alanine
Why do *second messenger systems* amplify signals?
because enzymes can catalyze a reaction more than once when they are active, and often activate other enzymes
When are conformational changes possible during a reaction according to the *Induced Fit* model of enzyme action?
before, during, and after the actual reaction, and can affect both the substrate and the enzyme ❗️in contrast with *Lock & Key* 🔐which claims it's only possible after a reaction
Triacylglycerols travel _____________ in the bloodstream between the liver and adipose tissue.
bidirectionally
What type of processes are *integrins* involved in?
bind & communicate with *extracellular matrix* *cell signalling* ⟶ cell division, apoptosis, attraction of platelets for blood clotting, WBC migration, stabilization of epithelium on basement memebrane, etc.
What are *positive control* mechanisms? Name an example.
binding of a molecule *increases* transcription of a gene. Ex: lac operon, specifically the binding of CAP to the promoter
What event switches a *GPCR* to the active state, initiating the intracellular signaling pathway?
binding of the heterotrimeric *G protein* after initial ligand binding to the GPCR
Vitamin B₇
biotin
Name the corresponding enzyme that joins *Okazaki fragments* for both prokaryotes and eukaryotes.
both *DNA ligase* ✴︎ Seals breaks in the DNA backbone between 3'OH and 5' PO4; requires energy source
Name the corresponding enzyme that synthesizes RNA primers for both prokaryotes and eukaryotes.
both *primase*
What happens to *Vmax* and *Km* during *uncompetitive inhibition*?
both are decreased at the same rate (therefore, slope stays the same!!!!) Explanation: *Vmax will be reduced* even though the E-S binding is enhanced because there are E-S-I complexes being formed which inhibit the formation of the product (remember, inhibitor "locks" 🔐the substrate). *Km decreases* because the inhibitor creates better E-S binding because it only binds to E-S complex. *But* the E-S complex is constantly being depleted as the inhibitor binds, producing E-S-I complexes. Therefore, to maintain the equilibrium between E-S and E-S-I complexes (following Le Chatelier's Principle), the reaction shifts toward *more* E-S formation where it will bind more substrate to the enzymes to create more E-S. Ultimately, this leads to a lower Km (*greater* affinity).
Why are CO and NO so toxic?
both of these molecules have a higher affinity for heme Fe₂²⁺ than oxygen, and so can *displace O₂* from hemoglobin.
What common purpose do glycerophospholipids and sphingolipids share?
both sites of biological recognition at the cell surface
When the *G protein* is inactive, where is the α-subunit?
bound to *GDP* and in a complex with the β and ɣ subunits
How do hydrolytic enzymes such as trypsin and chyrmotrypsin catalyze hydrolysis?
break apart the amide bond by adding a H to the amide nitrogen and an OH to the carbonyl carbon
What does *tRNA* do?
brings in AA and recognizes the codon on mRNA using its own *anticodon*
How are the various glycerophospholipids named?
by *head* group it can be polar, nonpolar, or neutral Explanation: it determines membrane surface properties
How can phospholipids be classified?
by backbone: ① Glycerol backbone ￫ *glycerophospholipid* ② Sphingosine backbone ￫ *sphingolipid*
What type of DNA is used in *DNA libraries*?
cDNA
The carboxylation of biomolecules usually provides...
calcium-binding sites Ex: prothrombin
*Cadherins* are a group of *glycoproteins* that mediate _____________ cell adhesion.
calcium-dependent
What is the risk with randomly integrated DNA during gene therapy with a viral vector?
can integrate near and activate a host oncogene ✴︎Case-and-point: small # of children treated for SCID developed leukemias
Chemically, why are triacylglycerols preferred storage compared to glycogen?
carbons are more reduced ￫ larger energy yield per unit weight plus, being hydrophobic means they do not need to carry extra weight from hydration layer also provides insulation ❗️one negative is that it takes longer to mobilize for energy compared to glycogen
In soap micelles, hydrophobic fatty acid tails face toward the center and ___________ groups face outward toward the water.
carboxylate (see top structure, the others are different detergents)
What is an *esterification* reaction?
carboxylic acid + alcohol ⟶ ester + water H+ catalyst
What could cause *mutarotation* to occur more rapidly?
catalysis with acid or base
How are *hydrolases* different than *lyases*?
catalyze the cleavage of molecules *using water*
What type of proteins do oncogenes primarily encode?
cell cycle-related proteins
What is the ABO blood typing system based on?
cell-surface antigens on RBC's ✴︎ these antigens are *sphingolipids*
What is the simplest sphingolipid?
ceramide
What molecules assist in the protein folding process by holding the protein in its tertiary structure?
chaperones **Hence, they are aptly named!
Unlike transferases, which always preserve both initial compounds in a reaction, enzymes that ____________ can eliminate some products.
cleave molecules
*Transferases* often use _____________ donors to move functional groups between molecules.
coenzyme ex of coenzymes: coenzyme A
What is the relationship between *coenzymes* and *cofactors*?
coenzymes ⇨ organic 🍎💊 cofactors ⇨ inorganic 🏆⚖️
*Oxidoreductases* typically use ____________ to facilitation redox reactions.
cofactors ex of cofactors: NAD+ and heme
Which protein makes up most of the extracellular matrix of connective tissue?
collagen
Inducible systems operate on a principle analogous to what type of reversible enzyme inhibition?
competitive Explanation: as the concentration of *inducer* increases, it will pull more copies of the *repressor* off the operator, freeing up those genes for transcription. So the inducer is like the inhibitor The repressor is like the active site on the enzyme And the operator is like the substrate. The inducer is also attracted to the repressor and competes with the operator for binding
What type of isomers are *leucine* and *isoleucine*?
constitutional
How are *restriction enzymes* used in *gene therapy*?
cut out a gene of interest from the *donor DNA* so that it can be introduced into a viral vector, which also must be cleaved to create an opening. then the two are merged together with help of *DNA ligase*
Vitamin B₁₂
cyanocobalamin
Where is mRNA translated into protein?
cytoplasm (ribosomes)
List the *pyrimidine* bases.
cytosine, uracil, thymine Mnemonic: *CUT* the *PY*e and pies only have *one ring* of crust
What is the unit typically used to express protein atomic mass for analytic purposes?
daltons (*Da*)
What enzyme removes oligosaccharides from a branch in glycogen or starches?
debranching enzyme
On a *Lineweaver Burke plot*, as you go up the y-axis, what happens to velocity of the reaction?
decreases
What kind of reaction is *glycoside bond* formation?
dehydration Explanation: ① If a sugar + lone alcohol: alkoxy ion replaces the OH from one molecule, which combines with a H from alcohol forming a leaving group (water) ② If 2 monosaccharides: OH from one sugar combines with H from the other, water leaves, left with the glycosidic bond
What lends to the extra stability of aromatic compounds?
delocalized π electrons Explanation: they can travel throughout the entire compound using available unhybridized p molecular orbitals
What is the difference between *ribose* and *deoxyribose*?
deoxyribose has an -H at the C-2' position, whereas ribose has an -OH there *an oxygen has been removed* ⟶ *de*oxy
terpenoids (isoprenoids)
derivatives of terpenes that have undergone oxygenation or rearrangement of the carbon skeleton similar function to terpenes (precursor, aromatic) named analogously to terpenes
What is *alternative splicing*?
different portions of an mRNA can be selected for use as exons. This allows either of two (or more) mRNA molecules to be made from one pre-mRNA. Alternative splicing is *not* a random process. Instead, it's typically controlled by regulatory proteins. The proteins bind to specific sites on the pre-mRNA and "tell" the splicing factors which exons should be used. Different cell types may express different regulatory proteins, so different exon combinations can be used in each cell type, leading to the production of different proteins
Which form of *Receptor Tyrosine Kinases (RTK)* is the active form?
dimer Explanation: starts off as a monomer that dimerizes upon ligand binding. The dimer is what *phosphorylates* additional cellular enzymes and *itself* (*autophosphorylation*).
Glycosylation of biomolecules usually serves what purpose?
directs cellular destination as proteins pass thru the ER and Golgi app
What happens to the ribosome after the polypeptide is released?
dissociates into its individual subunits, and also releases the mRNA template
What type of attractions hold the heavy and light chains together on an antibody?
disulfide linkages & noncovalent interactions
Vitamin A is what kind of terpene?
diterpene
Bacterial chromosome are (double/single) stranded DNA?
double, but *circular*
After each cycle of the *polymerase chain reaction*, what happens to the amount of DNA of interest?
doubles
Nucleotides that come after the initiation site are marked with positive numbers and said to be _____________.
downstream
When is the *spliceosome* formed?
during transcription
In neurons, ____________ bring vesicles of waste or recycled neurotransmitter to the negative end of the microtubule (toward the *soma*) through *retrograde transport.
dyneins
____________ are proteins involved in the sliding movement of cilia and flagella.
dyneins
With *ungated potassium channels*, there is a net __________ of potassium ions (when *not* at equilibrium).
efflux Explanation: because there is more inside, so it flows down gradient to *outside*
*Electrophoresis* uses what type of cell?
electrolytic ΔG > 0, Ecell < 0 so a *battery* is needed to drive this nonspontaneous process
What does *X-ray crystallography* specifically measure?
electron density
What is the advantage to *SDS-PAGE*?
eliminates conflation from proteins with similar mass-to-charge ratios because now they are only analyzed by *mass*
Another way to say *racemization* is...
enantiomerization
The D and L conventions of labeling sugars means that the two molecules are ____________.
enantiomers
*Restriction enzymes* are types of...
endonucleases
What molecules are responsible for different gene expression in different parts of the body?
enhancers and silencers, i.e. *tissue-specific* enhancers and silencers
What type of receptors are *Receptor Tyrosine Kinases (RTK)*?
enzyme-linked receptors
What type of receptors are *Serine/Threonine-Specific Protein Kinases*?
enzyme-linked receptors
Which of the following receptors displays *autoactivity*: - enzyme-linked receptors - G Protein-Coupled Receptors (GPCR)
enzyme-linked receptors Explanation: they function as receptors *and* have a catalytic domain, whereas the GPCR dissociates upon activation and transmits signals to an effector cell (but is not itself the effector).
How do the chemical properties of epimers compare? the physical?
epimers have *different* chemical AND physical properties
*Keratins* are intermediate filament proteins found in ___________ cells.
epithelial
What bonds are broken during saponification?
ester bonds of triacylglycerols are broken to form a glycerol molecule and salts of fatty acids (soap)
Which poses the most concern with regards to *gene sequencing*: ethics, physical risks, or inaccuracy?
ethics esp. *consent* and *privacy* with respect to relatives of those who were screened. There are no significant physical risks, and gene sequencing is fairly accurate.
*cDNA* contains only __________ of genes.
exons (transcriptionally active!) Explanation: cDNA lacks noncoding regions
What is RNA interference?
expression of a target gene is effectively silenced or knocked down by the selective inactivation of its corresponding mRNA by double-stranded RNA (dsRNA). RNAi is activated by dsRNA species delivered to the cytoplasm of cells. The silencing mechanisms can either lead to the *degradation* of a target mRNA, as induced by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs), or the *suppression* of translation of specific mRNAs, as induced by microRNA (miRNA)
Why are *sticky ends* advantageous in DNA technology?
facilitate the recombination of a restriction fragment with the vector DNA.
How reactive are *aromatic* compounds?
fairly *unreactive* because the delocalization of π electrons makes them stable
In *chromatography*, components with a high affinity for the mobile phase will migrate at what speed?
fast Explanation: if similar to the mobile phase (ex: both are polar), it will *catch the wave* and ride along quickly ❗️careful, with *ion-exchange* chromatography, the charged beads attract compounds with *opposite* charges.
How are micelles produced in the body?
fatty acids and bile salts secreted by the gallbladder form micelles that can increase the surface area available for lipolytic enzymes
How is *amylopectin* broken down and used for energy?
first needs to be processed by *debranching enzyme*, then follows amylose digestion
Vitamin B₉
folic acid
How are activity levels for enzymatic samples determined?
following the process of a known reaction, often accompanied by a color change ✴︎here, *activity* is referring to moles of substrate converted per unit time
soap is made up of...
free fatty acid salt
What are *snRNPs*?
function in splicing snRNA + proteins = snRNPs Details: - snRNA synthesized in nucleus by RNA polymerase II during transcription, given a normal 5' cap - exported to cytoplasm, where they complex with proteins ⟶ snRNPs - 5' cap gains 2 methyl groups, then they head back to the nucleus where they play a role in *splicing*
Lactose
galactose-β-1,4-glucose
What are the most complex sphingolipids?
gangliosides
What is the purpose of adding *dideoxyribonucleotides* during DNA sequencing?
given that they have a -H at C-3' versus a -OH, once one of these modified bases has been incorporated, the polymerase *can no longer add to the chain* *Allows for many fragments that all end with one of the modified bases that can then be separated by size using gel electrophoresis.* It's easy because the last base for each fragment can be read in order of size because that's how gel electrophoresis separates the strands! *Note*, each is labeled with a diff. color
Sucrose
glucose-α-1,2-fructose
Which is more rapidly mobilized for fuel, glycogen or triacylglycerols?
glycogen
Which two polysaccharides share all of their glycosidic linkage types in common? Cellulose and Amylopectin Amylose and Glycogen Amylose and Cellulose Glycogen and Amylopectin
glycogen & amylopectin Explanation: these are the only two polysaccharides that demonstrate branching, and thus have both α-1,6 and α-1,4 linkages.
gangliosides
glycolipids with polar head groups of oligosaccharides with one or more NANA molecules at the terminus. *negative* charge cell interaction, recognition, signal transduction Mnemonic: *gangly* = most complex structure and functional groups in all directions
cerebrosides
glycosphingolipids with one sugar
globosides
glycosphingolipids with two or more sugars
What happens to a titration curve when the pH is close to the pKa value of the solute?
goes relatively flat (see blue boxes) Explanation: *the solution is acting as a buffer*
The *Bradford protein assay* detects proteins with molecular weight of...
greater than 3-5 kDa
What is used to denature DNA?
heat, *alkaline* pH, chemicals (formaldehyde, urea, etc.)
Which enzyme is responsible for unwinding DNA?
helicase
How are *acetals* formed?
hemiacetal + alcohol under acidic conditions reaction is same to form ketals
How are *glycosidic bonds* formed?
hemiacetal sugar + alcohol under acidic conditions Note, in the context of linking sugars together, the alcohol group will simply come from another monomeric sugar. Shown is just ethanol. resulting C-O bond is the *glycosidic bond* new molecule (acetal) is called a *glycoside* this is a *dehydration* reaction reaction can be done with hemiketals, forming ketals
What is the difference between a *hemiacetal* and an *acetal*?
hemiacetal ￫ a carbon bonded to 2 oxygens, one of which is an OH, the other is not acetal ￫ carbon bonded to 2 oxygens, both of which are OR. Formed from reacting a hemiacetal + alcohol
Cyclic monosaccharides are...
hemiacetals or hemiketals
*Centromeres* are composed of what type of chromatin?
heterochromatin ⁂ like telomeres, also high GC-content
colloid
heterogeneous mixture whose particles never settle (undissolved) ex: adding soap to oil in water forms micelles and the overall mixture is a colloid
The *R state* of subunits/enzymes is characterized by __________ affinity.
high Mnemonic: *R* for *r*elaxed. When you're relaxed, you're *r*eceptive to binding with another molecule. Note, the conversion to the R state is only favorable when the right substrate comes along. For example, if there is no O₂ present, the T state is actually more stable and this lack of substrate encourages the transition from the *R* state ⟶ *T* state
When *sequestration* of a molecule is the goal, the *binding protein* usually has what degree of affinity for its target?
high & across a large range of concentrations Explanation: so that it can keep it bound at nearly 100%
Under what conditions might *Z-DNA* form?
high GC-content or high salt concentration
What conditions allow for high specificity of the hybridization probe to the target sequence?
high hybridization temperature or decreasing salt concentration
How does the free energy change from an unfolded protein to a folded protein?
high to low Explanation: protein folding is almost entirely driven by *entropy*✴︎ not enthalpy✿ Hydrophobic domains of a protein constrain the possible configurations of surrounding water✴︎✴︎, and so their burial upon folding *increases* the water's entropy (because now it can spread out/disperse more ⟶ greater entropy). ✴︎✴︎When hydrophobic portions *are* exposed to water (as they are when unfolded and not hidden in the interior) the water *cannot* interact with it, so the water interacts with nearby water molecules to rearrange themselves into neat arrangements that maximize hydrogen bonding. Sounds good? WELL IT'S NOT, because that's orderly, and the universe likes *disorder* not order. So ΔS < 0 here and ΔG > 0 (nonspontaneous). By moving the hydrophilic portions to the outside and *hiding* the hydrophobic portions, the water can *spread out* because it's not forced into these neat arrangements anymore. ✿Moreover, it turns out that hydrogen bonding of polar residues and the backbone is satisfied both in an unfolded state (by water) and in a folded state (by each other). Therefore enthalpy is "zero sum," and protein folding is driven almost entirely by entropy. *YOU MUST CONSIDER THE ENERGY OF THE PROTEIN ANDDDD THE SOLVENT* Folded = greater entropy overall Unfolded = less entropy overall
Does *DNA polymerase III* have high or low processivity?
high!
What type of RNA does RNA polymerase II transcribe?
hnRNA (pre-processed mRNA) some snRNAs
At high temperatures, cholesterol...
holds membrane intact and prevents it from becoming too permeable thus, it *makes it more solid*
What is an enzyme called if all necessary cofactors and coenzymes are present?
holoenzyme
What do *cellulose*, *starch*, and *glycogen* all have in common?
homopolysaccharides (all glucose) just different configuration @ anomeric carbon & position of glycosidic bonds
What is DNA *hybridization*?
https://ibb.co/bQYWtp joining of complementary base pair sequences using single-stranded sequences, creating either DNA-DNA or DNA-RNA recognition.
What is the role of *release factors* in translation?
https://ibb.co/bRbPUU *Termination* begins when a termination (stop) codon moves into the *A site* on the ribosome. BUT THERE IS NO tRNA w/ ANTICODONS THAT CAN PAIR WITH STOP CODONS! So what binds to the ribosome instead of tRNA? *release factors* Function ￫ they cause *peptidyltransferase* to hydrolyze the bond b/w the peptide chain and tRNA, thus releasing the polypeptide from the ribosome. The ribosome then dissociates into its individual subunits, releasing the mRNA
How do you convert a Haworth projection to a chair?
https://ibb.co/bXxPJ0
AMP binds to what residue on *DNA ligase*?
https://ibb.co/bYEhzU lysine
How can translation be *inhibited*?
https://ibb.co/bYGuDp miRNAs ￫ reduce the expression of their target genes, but they may play this role to produce many different outcomes (i.e. may be protective or lead to disease) Mechanism: ① A *miRNA* is first transcribed as a long RNA molecule, which forms base pairs with itself and folds over to make a hairpin. ② Next, the hairpin is chopped up by enzymes, releasing a small *double-stranded* fragment of about 22 nucleotides. One of the strands in this fragment is the mature miRNA, which binds to a specific protein to make an RNA-protein complex ③ The miRNA directs the protein complex to "matching" mRNA molecules (ones that form base pairs with the miRNA). When the RNA-protein complex binds: ￫ If the miRNA and its target match perfectly, an enzyme in the RNA-protein complex will typically chop the mRNA in half, leading to its breakdown. ￫ If the miRNA and its target have some mismatches, the RNA-protein complex may instead bind to the mRNA and keep it from being translated
What are the two types of *DNA libraries*?
https://ibb.co/cY1jDp 1. *Genomic* DNA libraries 2. *cDNA* (expression) libraries
What is the mechanism of action for *DNA ligase*?
https://ibb.co/cfFSYp Requires an input of energy (ATP in case of eukaryotes and NAD+ for prokaryotes). ① Splits the high-energy molecule (both result in AMP w/ different by-products) and forms an enzyme-AMP complex. ② The complex then binds to the nick that must expose a 5′ phosphate and 3' OH group. ③ The AMP then reacts with the phosphate group. ④ The 3' OH group on the moiety then generates a new phosphodiester bond, which seals the nick
What is a *probe* in the context of DNA analysis?
https://ibb.co/d2MsYp a fragment of DNA or RNA of variable length which can be radioactively labeled. It can then be used in DNA or RNA samples to detect the presence of nucleotide sequences (the DNA target) that are complementary to the sequence in the probe
List the 3 *posttranscriptional modifications* that occur during processing of hnRNA into mature *mRNA*. ✷✷✷You should remember that this *only occurs in eukaryotes*. In prokaryotes, it's basically ready to be translated right after transcription!
https://ibb.co/dcnxip Before actual processing, protein complexes are formed: (a) *hnRNA* (mostly pre-mRNA) is rapidly covered by proteins to form heterogenous nuclear riboproteins (hnRNPs). *hnRNP proteins* stabilize *hnRNA* (most of which is pre-mRNA), keeping it in a single-strand form and assisting in various processing functions, which include... ① *5' cap is added* (remember, this actually happens during transcription, shortly after initation and while elongation is still in process). ￫ this feature is recognized by the ribosome as the binding site ￫ *protects mRNA* from degradation in the cytoplasm ￫ splicing of the first *exon* ② a *polyadenosyl (poly-A) tail* is added by *poly(A) polymerase* to the 3' end *after the polyadenylation signal & cleavage of the primary transcript*, so it is indeed posttranscriptional. ￫ composed of *adenine* bases that can be > 200 nucleotides long ￫ the tail is a protein-binding site that protects against rapid degradation and export out of the nucleus ￫ splicing of last *intron* ③ *splicing* ￫ in the *spliceosome*, *snRNA* (another type of *hnRNA*) complex with proteins to form *snRNPs* The formation of the spliceosome happens *during transcription* ￫ snRNPs recognize both the 5' and 3' splice sites of the *introns*. ￫ These noncoding sequences are excised in the form of a *lariat* ￫ exons are *ligated* together
Describe the process of *gene therapy* in humans.
https://ibb.co/ezpJ69 ① *Modify* a virus to use as a vector ￫ replace portion of viral genome with *cloned* gene so that virus can infect but *not* complete its replication cycle ② Multiple virions carrying the therapeutic gene produced ③ integration of virus (not able to replicate) so that gene is now apart of the host DNA ④ Host machinery creates the therapeutic gene product with this new information
List the steps in *translation*.
https://ibb.co/fUrSzU ① Initiation (only step where different b/w prokaryotes and eukaryotes!) *Eukaryotic Initiation*: (a) Methionyl-tRNA forms a complex with *eIF2*, which binds *GTP* (b) This complex then binds to the small ribosomal subunit (*40S*) with the help of *eIF3* (this IF also helps prevent premature association of the 60S ribosomal subunit w/ this preinitiation complex) (c) 5' cap of the mRNA binds to a *eIF4* complex ("cap-binding complex") (d) The *preinitiation complex* (Met-tRNA + 40S + IF's + GTP) attaches to the other complex formed in step c (*5' cap* of the mRNA + *eIF4* complex) (e) Unwinding of a *hairpin loop* and scanning for a *start codon* (happens via a eIF *helicase* reaction that requires ATP) within the *Kozak sequence* (which is upstream of the AUG start codon) (f) When it locates AUG, - *GTP* is hydrolyzed - *IF*'s are released - Large ribosomal subunit (*60S*) binds *Ribosome is now complete* (g) 40S lays down *methionine* in the *P site* of the ribosome, located initially at the start codon for translation. *Prokaryotic Initiation*: (a) Formyl-methionyl-tRNA forms a complex with only 3 *IF*s. (b) *Shine-Dalgarno sequence* (upstream of AUG) binds to the complementary sequence near the 3' end of a portion (16S) of the *30S* small ribosomal subunit and scans for the AUG *start codon*. (b) subunit lays down *N-formylmethionine* in the *P site* of the ribosome. ------------------------------------------------ ② Elongation (a) Addition of a new aminoacyl-tRNA into the *A site* of the ribosome - GTP is hydrolyzed to GDP and more GTP is quickly replaced by *elongation factors* (EFs) (b) Formation of a *peptide bond*: transfer of the growing polypeptide chain from the tRNA in the *P site* to tRNA in the *A site* (c) Translocation: (d) Ejection of the free tRNA: the now *uncharged* (empty) tRNA pauses in the *E site* before *e*xiting the ribosome ------------------------------------------------ ③ Termination (a) occurs when the codon in the *A site* is a *stop codon*. (b) a *release factor* places a water molecule on the polypeptide chain ⟶ releasing the protein ✴︎ There are copious initiation factors (IF), elongation factors (EF), and termination factors (TF) that were not mentioned that assist in each step along the way by recruitment, assembly, and disassembly of the ribosome *END OF TRANSLATION* Followed by *posttranslational modifications* in both prokaryotes and eukaryotes
What is required for *PCR*?
https://ibb.co/fZNAKU ① template DNA strand ￫ what polymerase reads ② *heat* ￫ denatures the double helix ③ *primers* that are complementary to the DNA that flanks the region of interest (a/k/a the DNA on both sides of the piece you want); has high GC content ￫ gives polymerase a place to start, just like in DNA replication ④ nucleotides (dATP, dTTP, dCTP, and dGTP) ￫ this is what Taq polymerase adds to the primer and creates the large amount of copies ⑤ Taq polymerase ￫ does the copying
What are the various ways in which translation is regulated?
https://ibb.co/g0fAm9 ① During *initiation* ￫ the formation of the ribosome complex involves many *helper proteins (IFs) that make sure everything is correctly positioned. ex: in order for translation to begin, *eIF-2* must bind to the small ribosomal subunit. Binding of eIF-2 is *controlled by phosphorylation*. - When eIF-2 is phosphorylated, it's turned "off", it undergoes a shape change and can no longer play its role in initiation, so translation cannot begin. - When eIF-2 is not phosphorylated, it's "on" and can carry out its role in initiation, allowing translation to proceed. In this way, phosphorylation of eIF-2 acts as a switch, turning translation on or off. *Inactivation of translation can be a good strategy in periods when the cell can't "afford" to make new proteins* ② *After* translation: I. Chaperone folding II. Formation of 4° structure III. Cleavage of proteins or signal sequences (e.g. insulin must be proteolytically cleaved to become active) IV. Covalent addition, including but not limited to... (a) Phosphorylation (b) Ubiquitination (c) Methylation (d) Acetylation etc.
What are *repressible* systems? Name an example.
https://ibb.co/g5PHzU These systems are transcribed ("on") under normal conditions. The *repressor* is turned off, allowing transcription. However, the system can be turned off by a *corepressor* coupling with the repressor (turning it *on*) and the subsequent binding of this complex to the operator. Ex: *trp operon* This operon encodes 5 enzymes required for the synthesis of *tryptophan*. When Trp is available, E. coli save energy by no longer making these enzymes (they don't need to!) Trp is the *corepressor* that binds to the inactive repressor, causing a conformation change and binding to the operator. Thus, transcription is inhibited Sound familiar? ⟶ *negative feedback*
Describe the steps of *Southern blotting*.
https://ibb.co/gKsXYp ① DNA cut by restriction enzymes ② fragments separated by gel electrophoresis ③ fragments transferred by buffer capillary action to a *nitrocellulose membrane*, retaining their separation. Incubated to ensure a good stick ④ membrane probed w/ many copies of a *single-stranded* DNA sequence. *Probe* binds its complementary sequence and forms *double-stranded* DNA (*hybridization*). Use detergent like SDS to ensure *specific* binding ⑥ After *hybridization*, excess probe washed off membrane, and pattern viewed on X-ray via *autoradiography* (if probe was fluorescent/radioactive) or development of color if chromogenic method used.
What is *telomerase*'s mechanism of action?
https://ibb.co/gjJWtp The telomerases contain an integral *RNA component* which has a sequence complementary to the repeat sequence and this acts as a template for a reverse transcriptase reaction. The telomerase then moves to the new 3' end once the repeat has been added and does it all again. The telomerase *protein component* is a type of *reverse transcriptase*. The other strand then copies the sequence by normal lagging strand mechanisms. The 3' end of telomeres has an overhang and this mechanism accounts for this.
What must a *DNA vector* contain in order to be used for recombinant DNA technology?
https://ibb.co/h0b6R9 ① at least one sequence recognized by restriction enzymes ② an origin of replication ③ at least one gene for *antibiotic resistance*
What is *shRNA* and how is it different from *siRNA*?
https://ibb.co/hUULop Short hairpin RNA ⟶ a useful biotechnology tool used in *RNA interference* (RNAᵢ). Like siRNA, can be used for protein knockdown by gene silencing. They are converted into siRNAs by the same RNAᵢ machinery that processes miRNAs. The difference is in how they are loaded into the protein: shRNA is *vector-based* ⟶ introduced into the nuclei of target cells using vectors that can sometimes stably integrate in the genome. Once loaded into the protein (RISC), the process of target mRNA recognition and degradation by both shRNA and siRNA is essentially the same. *Advantages of shRNA over siRNA* include: - the ability to use viral vectors for delivery to overcome the difficulty of transfecting certain cell types - the option to control shRNA expression using *inducible promoters* -the ability to *co-express* them with a reporter gene. - Additionally, they may cause fewer off-target effects.
Once the transcription complex is formed, basal transcription begins and maintains moderate, but adequate levels of the protein encoded by this gene in the cell. There are times, however, when the expression must be increased (*amplified*) in response to specific signals, such as *hormones*, *growth factors*, and other intracellular conditions. How do eukaryotic cells accomplish this?
https://ibb.co/hwEjw9 Gene amplification occurs in two ways: ① *Enhancers* ￫ made up of *signal molecules* *transcription factors* and *response elements* By utilized enhancer regions, which are much further upstream from the gene than promoters, genes have an increased likelihood to be amplified bc of the variety of signals that can increase transcription levels. *Enhancers allow the same gene to be used in diverse processes in space and time* ② Gene duplication ￫ the relevant gene is duplicated either in series on the same chromosome *or* in parallel by opening the gene with helicases and permitting DNA replication only of that gene Cells can continue replicating the gene until 100s of copies of the gene exist in parallel on the same chromosome
How are a codon and anticodon lined up?
https://ibb.co/iEqJ69 antiparallel See link for a sample question & make sure you got it down
How is *prokaryotic translation* different from *eukaryotic translation*?
https://ibb.co/iQqF3p Only in the *initiation step* Prokaryotes: - First AA: *formyl-methionyl-tRNA* (formyl-methionine) - Initiation Factors (IF's): only 3 - Binding of mRNA to small ribosomal subunit is different (*since prokaryotic mRNA is NOT capped*): *Shine-Dalgarno sequence* upstream of initiating AUG binds to complementary sequence in *16S* rRNA - Difference in ribosome *size* ￫ 70S ribosomes, made up of a 50S (large) & 30S (small) *Note*, they do not add up! Eukaryotes: - First AA: *methionyl-tRNA* (methionine) - euk Initiation Factors (eIF's): 12+ - Binding of mRNA to small ribosomal subunit is different: *5'-cap* of mRNA binds *eIF*s and 40S + met-tRNA complex. THEN, mRNA is scanned for AUG *within the Kozak consensus sequence* - Difference in ribosome *size* ￫ 80S ribosomes, made up of a 60S (large) & 40S (small) *Again*, they do not add up!
What are the two types of *termination signals* that end elongation during transcription in *prokaryotes*?
https://ibb.co/ipMFm9 ① hairpin loop forms in the transcript, preceding several U residues ② rho (ρ) factor protein binds ￫ release of RNA transcript from the templates (*requires* energy)
Describe the process of *DNA cloning*.
https://ibb.co/iwUbR9 ① ligate DNA of interest into a vector, forming a *recombinant vector* ② Transfer vector to a host bacterium ③ Grow bacteria in colonies ④ Isolate colony containing recombinant vector ⑤ Grow this colony in large quantities ⑥ Next step there are *two options*: A. Make bacteria *express* gene of interest by generating large quantities of recombinant protein. *End result*: recombinant *proteins* or B. Lyse bacteria to reisolate plasmid vectors, then expose them to restriction enzymes that will release the cloned DNA from the vector. *End result*: large amounts of cloned, homogenous *DNA*
What are the steps to producing a *knockout mouse*?
https://ibb.co/iwvEeU ① Isolate knockout gene from a mouse library. ② New DNA sequence is engineered which is very similar to the original gene and its immediate neighbor sequence, except it's changed enough that the new gene is inoperable. Usually, the new sequence is also given a marker gene, (a gene that normal mice don't have that confers resistance to a certain toxic agent or that produces an observable change, e.g. color or fluorescence). In addition, a second gene is also included to accomplish a complete selection. ③ Embryonic stem cells are isolated from a mouse *blastocyst* (a very young embryo) and grown in vitro. For this example, we will take stem cells from a white mouse. ④ The new sequence from step 2 is introduced into the stem cells from step 3 by *electroporation*. By the natural process of *homologous recombination*, some of the electroporated stem cells will incorporate the new sequence with the knocked-out gene into their chromosomes in place of the original gene. The chances of a successful recombination event are relatively low, so the majority of altered cells will have the new sequence in only one of the two relevant chromosomes (heterozygous). Cells that were transformed with a vector containing the neomycin resistance gene and the herpes tk+ gene are grown in a solution containing neomycin and Ganciclovir in order to select for the transformations that occurred via homologous recombination. Any insertion of DNA that occurred via random insertion will die because they test positive for both the neomycin resistance gene and the herpes tk+ gene, whose gene product reacts with Ganciclovir to produce a deadly toxin. Moreover, cells that do not integrate any of the genetic material test negative for both genes and therefore die as a result of poisoning with neomycin. ⑤ The embryonic stem cells that incorporated the knocked-out gene are isolated from the unaltered cells using the marker gene from step 2. For example, the unaltered cells can be killed using a toxic agent to which the altered cells are resistant. ⑥ The knocked-out embryonic stem cells from step 5 are inserted into a mouse blastocyst. For this example, we use blastocysts from a grey mouse. The blastocysts now contain two types of stem cells: the original ones (from the grey mouse), and the knocked-out cells (from the white mouse). ⑦ These blastocysts are then implanted into the uterus of female mice, where they develop. The newborn mice will therefore be chimeras: some parts of their bodies result from the original stem cells, other parts from the knocked-out stem cells. Their fur will show patches of white and grey, with white patches derived from the knocked-out stem cells and grey patches from the recipient blastocyst. Some of the newborn chimera mice will have gonads derived from knocked-out stem cells, and will therefore produce eggs or sperm containing the knocked-out gene. When these chimera mice are crossbred with others of the wild type, some of their offspring will have one copy of the knocked-out gene in all their cells. These mice will be entirely white and are not chimeras, however they are still heterozygous. When these heterozygous offspring are interbred, some of their offspring will inherit the knocked-out gene from both parents; they carry no functional copy of the original unaltered gene (i.e. they are homozygous for that allele). *This is our knockout mouse*
List the steps of *prokaryotic* transcription.
https://ibb.co/kcXZeU ① Initiation: (a)*RNA polymerase II* combines with *sigma factor* to the *promoter* (TTGACA in the -35 region). (b) This causes the DNA double helix to unwind and separate within a region of ~ 10-20 nucleotides in length ② Elongation: (a) RNA polymerase "walks" along the *template strand* *3'⟶ 5'* and transcribes on the growing RNA chain *5'⟶3'* via addition of complementary nucleotides to the 3' end. As RNA polymerase transcribes the DNA, the untranscribed region of the helix continues to separate, while the already-transcribed region of the DNA template rejoins its DNA partner. (b) σ factor is released when the growing RNA chain is ~ 10 nucleotides long Elongation continues until... ③ Termination: RNA polymerase encounters one of 2 *transcription termination signals*, which themselves are signaled by the sequence of bases in the newly synthesized RNA. *END OF TRANSCRIPTION* Left with mRNA that is already being translated!
List the steps of *transcription* in eukaryotes
https://ibb.co/mpGJpU ① Initiation: (a) *transcription factors* & *hnRNP*s bind to the *TATA box* in the promoter of the gene (or *initiator* if no TATA box). This facilitates the binding of RNA polymerase II to the complex of transcription factors on one side and to DNA on the other side. (b) Three other transcription factors bind, *cleaving ATP*, and transcription is initiated (b) One of these transcription factors, TFIIH, acts as a *DNA helicase* unwinding DNA into single strands for transcription ✷ Note, it is the RNA polymerase-transcription factor *complex* that unwinds DNA, and it is acting like a helicase. That's why some people say helicase initiates and some say RNA polymerase but they are all one complex. ② Elongation: (a) the *template strand*, acts as a template for RNA polymerase. As it "reads" this template one base at a time, the polymerase builds an RNA molecule out of complementary nucleotides, making a chain that grows from *5' to 3'*. The *RNA transcript carries the same information as the non-template (coding) strand* of DNA, but it contains U instead of T (b) The already-transcribed region of the DNA template rejoins its DNA partner, while the untranscribed region continues to unwind and be transcribed. (c) σ factor released when the growing RNA chain is ~ 10 nucleotides long (d) A *7-methylguanylate triphosphate cap* is covalently added to the 5' end of the hnRNA molecule (note, this step is considered part of mRNA processing, but it actually occurs shortly after initiation and while elongation is still in process). (e) elongation continues until... RNA polymerase encounters a *polyadenylation signal*... ③ Termination: (a) RNA polymerase transcribes the stop codon and the passes a *polyadenylation signal*. (b) This signal is recognized by an enzyme that cuts the RNA transcript nearby, releasing it from RNA polymerase. RNA polymerase strangely continues past this signal until it reaches an unknown *termination signal* many nucleotides later after the transcript was released. (c) Eventually, RNA polymerase detaches from the DNA through mechanisms that are not yet fully understood. The extra RNA is not usually translated and seems to be a wasteful byproduct of transcription *END OF TRANSCRIPTION* We are left with a *primary transcript * of *hnRNA*, most of which is *pre-mRNA* which is what will eventually become mature mRNA. We have already undergone some *processing* - 5' cap But before we can proceed into *translation*, there's are two more *posttranscriptional modifications* that must take place... *POLY(A)-TAIL* *SPLICING*
What happens during the *elongation* phase of translation? ✴︎✴︎Remember, the mechanisms are very similar in prokaryotes and eukaryotes with the exception of different elongation factors, etc. ✴︎✴︎
https://ibb.co/msgNip (I) Addition of a new *aminoacyl-tRNA* into the *A site* of the ribosome - occurs when its *anticodon* is antiparallel & complementary to the mRNA codon. - GTP is hydrolyzed to GDP and more GTP is quickly replaced by *elongation factors* (EFs) so that another aa-tRNA can bind in the A site. (II) Formation of a peptide bond by *peptidyltransferase*: the AA on the tRNA in the A site forms a *peptide bond* with MET on the tRNA in the P site, transferring the growing polypeptide chain from *P site* ⟶ *A site*. The P site is now uncharged (no AA or peptide). (III) Translocation: eEF2 complexes with GTP and binds to ribosome ⟶ conformational change that *moves the mRNA & its base-paired tRNAs so that the next codon of mRNA occupies the A site* (it's a shift left/forward in the diagram). (IV) Ejection of the free tRNA: Also resulting from the conformational change, the now uncharged tRNA moves from the *P site ⟶ pauses in the *E site*. It is released from the ribosome *when the next charged tRNA enters the A site* (back to step 1 in the figure).
Pepsin is what type of enzyme?
hydrolase
*Phosphatase* falls under what class of enzymes?
hydrolases Explanation: cleaves a phosphate group from another molecule via addition of water
Out of the 6 classes of enzymes, two classes both *cleave molecules*. What are the names of these classes of enzymes?
hydrolases and lyases
What tendency prevents amino acid *sequencing* after complete protein hydrolysis and subsequent chromatographic analysis?
hydrolysis is random, so only the individual AA that compose the protein can be determined, *not* the order in which they were *sequenced* (*primary structure*)
What two functional groups on a monosaccharide allow it to undergo intramolecular hemiacetal/hemiketal formation reactions?
hydroxyl (-OH) = nucleophile carbonyl (C=O) = electrophile
Why is the *Lock and Key* hypothesis weakened by the concept of *competitive inhibition*?
if a competitor can bind, that means there's more than one "key" that can fit a particular lock.
What are the two ways that aldoses may get oxidized?
if in straight chain form: aldehyde ￫ *aldonic acid* or terminal hydroxyl ￫ *uronic acid* or both ￫ *aldaric acid*. No matter where oxidation occurs, a carboxylic acid is the result if in cyclic form: hemiacetal ￫ *lactone*
What makes a monosaccharide a *reducing sugar*?
if it's an *aldose*, it can be oxidized, therefore it's a reducing agent. So, if it has a *hemiacetal* ring, it can be a reducing sugar All monosaccharides by definition are not in glycosidic linkages so the hemiacetal is available
What could permit a *binding protein* involved in sequestration to have a low affinity for its substrate and still have a high percentage of substrate bound?
if the binding protein is *present in high quantities* relative to substrate, nearly all substrate will be bound despite a low affinity
What is the term for an aromatic ring with two nitrogen atoms?
imidazole Ex: histidine
Which enzyme is responsible for unwinding the DNA double helix in prokaryotes? In eukaryotes?
in both ￫ *helicase*
Where is the *TATA box* located? Is it found in *prokaryotes* or *eukaryotes*?
in the *promoter*, specifically ~ 25 base pairs *upstream* from first transcribed base *eukaryotes*
Why is the use of multiple *transcription factors* to regulate a single genei beneficial for efficient gene expression?
in this way, different sources of information can be integrated into a single outcome. For instance, imagine that: Activator A is present only in skin cells Activator B is active only in cells receiving growth factor signals from neighbors Repressor C is produced when a cell's DNA is damaged In this case, the gene would be "turned on" only in skin cells that are receiving division signals and have undamaged, healthy DNA. This pattern of regulation might make sense for a gene involved in cell division in skin cells. In fact, the loss of proteins similar to repressor C can lead to cancer (uncontrolled cell division!)
What effect does *adenylate cyclase* have on cAMP?
increases levels of cAMP
Why do prokaryotes have polycistronic mRNAs?
increases the *viability* of gene products from one transcript This type of transcript is *translated as it is being transcribed*. It is *not* modified and trimmed, and it does *not* contain introns W/ prokaryotic organisms, its sensible and economical that all functionally-related proteins are synthesized together since they are less complex.
How does *heat* denature proteins?
increases their average kinetic energy, thus disrupting hydrophobic interactions that hold the protein together, causing it to unfold
What happens to *Km* during competitive inhibition? (show on a Lineweaver Burk plot)
increases, because affinity is reduced
If 1/[s] = 0, [s] must be...
infinite (HUGEEEEE) This occurs at the *origin* on the graph
NSAIDs
inhibit COX, which aids in the production of *prostaglandins*
What is *noncompetitive inhibition*?
inhibitor binds to an *allosteric* site, not the active site, but it *does* change the conformation of the active site, preventing the substrate from binding.
All *CAMs* are _________ proteins.
integral membrane proteins
What type of proteins are *keratins*?
intermediate filament
What are *molten globules*?
intermediate states in the folding (3°) of a protein forms fast!
What is the role of *topoisomerases*?
introduce *negative* supercoils that relieve torsional pressure due to unzipping by helicase
What is the evolutionary importance of *introns*?
introns play a role in *alternative splicing* ￫ able to make a greater variety of proteins from a limited # of genes Thought to play roles in... - directing transcription - posttranscriptional regulation of gene expression levels Example: spliceosomal introns can both stimulate & repress transcription and also control transport of mRNA out of the nucleus - maintaining the size of our genome - allow for rapid protein evolution ￫ many euk. proteins share peptide sequences in common, suggesting that the genes encoding for these particular peptides may employ a *modular function* (they contain standard sequences that can be swapped in and out, depending on the needs of the cell) Currently, we have discovered *transposons* which show how genes can hide in introns and be expressed thru other mechanisms
How does iodine test for the presence of starch?
iodide ions fit inside the helix conformation of amylose, forming a starch-iodine complex where there is an *intermolecular transfer of charge* amylose will give a *blue* color, amylopectin will give a *purple* color
Out of the 6 classes of enzymes, two classes both *affect the bonding of molecules*. What are the names of these classes of enzymes?
isomerases and ligases
How does DNA accommodate for the large distance between *enhancer* and *promoter* regions on a given gene?
it bends into a *hairpin loop* to bring these elements together spatially
How does vitamin K modify prothrombin?
it has an aromatic ring that undergoes a cycle of oxidation and reduction
How does *Tollen's reagent* test for reducing sugars?
it itself is *reduced* and produces a silvery mirror when aldehydes are present The aldehyde can be *oxidized* https://ibb.co/bzoTVf
When *transport* of a molecule is the goal, the *binding protein* usually has what degree of affinity for its target?
it varies depending on environment Explanation: must be able to bind *or* unbind its target to maintain *steady-state* concentrations If concentration is low ￫ binding protein has a low affinity and releases it/doesn't pick it up in this region. It will go around to another area where the concentration is high and then *its affinity will change* (increase) so that it can pick it up and bring it back to where it is needed If concentration of the molecule is adequate/too high ￫ binding protein has high affinity to remove it from the pool and bring it to where it's needed.
What is inherently wrong with the phrase "cytokine DNA"?
it's a misleading term: cytokines, as proteins, don't have their own DNA!
What is the advantage of *PCR*?
it's automated, and can produce millions of copies of a DNA sequence *without* amplifying the DNA in bacteria
Why must a material like *nitrocellulose* be used as a membrane for DNA transfer in Southern blotting?
it's polar ￫ ion exchange interactions *bind the DNA to the membrane* due to the negative charge of the DNA and positive charge of the membrane
The E-S complex can dissociate back into E + S at a rate of k₋₁ or turn into E + P at a rate of _________.
kcat
At low temperatures, cholesterol...
keeps the cell membrane from solidifying thus, it *makes it more fluid*
Which form predominates at equilibrium, the keto or enol?
keto
D-fructose
ketohexose
What happens in *keto-enol tautomerization*?
ketone picks up a H while the double bond is moved b/w 2 adjacent carbons, resulting in an *enol*
In neurons, ____________ bring vesicles of the neurotransmitter to the positive end of the axonal microtubules (toward the *synaptic terminal*).
kinesins
___________ are proteins that play key roles in 1. aligning chromosomes during metaphase and 2. depolymerizing microtubules during anaphase of mitosis.
kinesins (both shown in pic)
Name an *inducible system*. What happens in such a system?
lac operon https://ibb.co/mCACYp Inducible systems are bonded to a *repressor* under normal conditions. They can be turned *on* by an *inducer*, which pulls the repressor off the operator on the operon. In the above example, the lac operon contains the gene for *lactase* which digests lactose. Bacteria normally prefer glucose, but if glucose is low and lactose is high, this operon will be *induced* with the help of *CAP* (a transcriptional activator). CAP is recruited when falling levels of glucose cause ↑ in *cAMP*, which binds to CAP. This causes a conformational change in CAP which allows it to bind to the *promoter* on the operon, further increasing transcription of lactase.
What type of molecule is vitamin C?
lactone (cyclic ester w/ carbonyl on anomeric carbon)
In *size-exclusion* chromatography, which molecules will pass through fastest?
large Explanation: small molecules can enter the tiny pores in the column, thus getting stuck
What is the first step in the replication of DNA?
lay down an RNA primer (by *primase*)
In *L-amino acids*, the amino group is drawn on the ___________ in a Fischer projection.
left
What is the usefulness of high GC-content in certain parts of the chromosome?
lends strength where it is needed via increased hydrogen bonding: ￫ *telomeres* need to be strong so the ends of the chromosome don't unravel ￫ *centromeres* need to keep the two sister chromatids connected until microtubules separate them during anaphase.
What happens during *mixed inhibition*?
like *noncompetitive* inhibition, but now the inhibitor does *not* have equal affinity for enzyme and enzyme substrate complex. Mixed inhibitors bind at an *allosteric* site, *not* the active site. So if the inhibitor prefers the enzyme ￫ acting like a competitive inhibitor, so apparent Km *increases* (lower affinity). and if it prefers the E-S complex ￫ acting like an uncompetitive inhibitor, so apparent Km *decreases* (higher affinity). Apparent Vmax will decrease in either case because the inhibitor binds the allosteric site, not the active site; thus, even if we increase the concentration of [S], the inhibitor can still always bind to this distinct site, so the velocity of the reaction will decrease no matter what
What organs convert vitamin D to its biologically active form?
liver and kidneys
What role do *elongation factors* play in translation? (Think big picture)
locating and recruiting aminoacyl-tRNA along with *GTP*, while helping to remove GDP once the energy has been used.
How do you determine if a sugar is *D* or *L*?
look at bottom (highest #) chiral carbon in the Fischer projection If the -OH is on the right ￫ D If the -OH is on the left ￫ L
How does DNA polymerase differentiate between the parent and daughter strand when *proofreading*?
looks at the level of *methylation*: more methylated = parent strand (it's older)
Cofactors and coenzymes are usually kept at (low/high) concentrations in cells.
low Explanation: so they can be recruited only when needed!
The *T state* of subunits/enzymes is characterized by __________ affinity.
low Mnemonic: *T* for *t*ense. When you're tense, you don't want to be *t*ouched by another molecule.
surfactant
lowers the surface tension at the surface of a liquid, serving as a detergent or emulsifier
Which RNA is the only type that contains information that is translated into protein
mRNA the others (rRNA and tRNA) *stay* as RNA each with their own specific functions
What does *rRNA* do?
makes up the ribosome and is *enzymatically* active
What would also classify a sphingolipid as a phospholipid?
many of them have *phosphodiester linkages* They would *NOT* be a phospholipid if they have glycosidic linkages to sugars
How does a *repressor* transcription factor work?
many ways, but as one example, a repressor may get in the way of the basal transcription factors or RNA polymerase, making it so they can't bind to the promoter or begin transcription
What functions do keratins perform?
mechanical integrity of the cell & also act as regulatory proteins
What is the primary *posttranscriptional modification* that transforms hnRNA to *r*RNA?
methylation **snRNPs are involved in pre-rRNA processing as they define these methylation sites, but we know that they are also *highly* involved in pre-mRNA processing!
What are the units of measurement for *Vmax*?
moles of enzyme per second
Do most eukaryotes have polycitronic or monocistronic mRNAs?
monocistronic Explanation: this type requires posttranscriptional processing, Can be started by *one* start codon and stopped by *one of three* stop codons The problem with the polycistronic genome is that all the structural genes of an operon are turned ON or OFF together, due to a single promoter and operator upstream to them. In case of eukaryotes, who are much more complex, often a single protein is involved in many processes. So, eukaryotes have evolved to have monocistronic genes.
In the *Bradford protein assay*, increased protein concentrations correspond to what color dye in solution?
more blue
What does it mean when the DNA double helix is called *B-DNA*?
most common form: it is a *right*-handed helix that makes a turn every *3.4 nm* and contains about *10* bases within that span
What type of RNA does RNA polymerase I transcribe?
most rRNA (28S, 18S, 5.8S)
Myosin is a __________ protein.
motor
The tertiary structure of a protein is primarily the result of...
moving the hydrophobic AA side chains into the interior of a protein
How many origins of replication do eukaryotes have?
multiple
What is meant by *degenerate code*?
multiple codons encode for the same AA Ex: stop codons ￫ UAA, UGA, UAG
What must happen to DNA before replication and transcription can occur?
must separate into two *single-strands*
*PAGE* analyzes proteins in their ___________ state.
native
*RNA* has what overall charge?
negative (-)
When Hill's coefficient < 1, what type of cooperative binding is occurring?
negative (red in picture) Explanation: after one ligand is bound, the affinity of the enzyme for further ligands decreases
*Electrophoresis* works by subjecting compounds to an electric field, which moves them according to...
net charge *and* size
Vitamin B₃
niacin
Is sucrose a reducing sugar?
no b/c the glycosidic linkage is b/w the two anomeric carbon atoms & thus it cannot convert to an open-chain form with an aldehyde group it's stuck in the cyclic form
Is *trehalose* a reducing sugar?
no b/c the glycosidic linkage is b/w the two anomeric carbon atoms (α,α-1,1) & thus it cannot convert to an open-chain form with an aldehyde group it's stuck in the cyclic form
What effect do *silent mutations* have on protein synthesis?
no effect
How is DNA technology used for *forensic pathology* and crime scene investigation?
noncoding regions help identify certain individuals Ex's: • STR's (*short-tandem repeats*) ￫ short sequences of DNA (2-6 base pairs long) that are normally found in very high amounts in different individuals • mitochondrial DNA (*mtDNA*) ￫ useful even if only a very little sample • Y chromosome typing (Y-STR) ￫ STR on the Y chromosome
_____________ inhibitors have equal affinity for both the enzyme and the enzyme-substrate complex.
noncompetitive
*SDS* disrupts all _____________ interactions.
noncovalent
When Hill's coefficient = 1, what type of cooperative binding is occurring?
none (blue in picture) Explanation: this enzyme does not exhibit any cooperative binding
In *column chromatography*, the mobile phase is ____________.
nonpolar solvent
In what context are *ligases* most often encountered?
nucleic acid synthesis and repair Explanation: DNA *ligase*!!!!!!!!
Besides proteins, what other molecules can *column* chromatography isolate and collect?
nucleic acids
*Adenosine* is a... A. base B. nucleoside C. nucleotide
nucleo*s*ide
Where is *rRNA* synthesized?
nucleolus
What comprises a *nucleotide*?
nucleoside + one or more phosphate groups attached to C-5', so: *sugar* + *base* + *phosphate group/s*
How are *thymine dimers* repaired?
nucleotide excision repair ① proteins scan the DNA and recognize a bulge ② *excision endonuclease* makes nicks in the phophodiester backbone of damaged strand on *both* sides of the T dimer and removes the defective oligonucleotide ③ DNA polymerase fills gap *5' ⟶ 3'* using the undamaged strand as a template ④ DNA ligase seals nick in the strand
Where is mRNA transcribed?
nucleus
Where are the binding sites for a *transcription factor*?
often close to the gene's promoter, however, they can be very far away and still affect transcription.
How does an *activator* transcription factor work?
often help an enzyme bind to the promote, stabilize complexes, etc.
How many origins of replication do prokaryotes have?
one origin thus, two replication forks
What is *uncompetitive inhibition*?
only binds to the enzyme-substrate complex, and not the enzyme alone So the substrate *can* bind to the active site. This *changes the conformation of the allosteric site so that the inhibitor can now bind*, and then it prevents the substrate from being released.
What are *chimeras*?
organisms that contain cells from two different lineages Ex: mice formed by integration of transgenic embryonic stem cells into a normal mouse blastocyst
In redox reactions, the electron *acceptor* is called a _____________.
oxidant (oxidizing agent) Explanation: it *oxidizes* the other molecule by stealing its electrons and is itself reduced.
What are some possible names for *oxidoreductases*?
oxidases, reductases, peroxidases, dehydrogenases, etc. ✴︎can be hard to identify, they are pretty ubiquitous. Tip: If you have no idea what type of enzyme you're dealing with, this is a good guess!
Vitamin B₅
pantothenic acid
What type of signalers are prostaglandins?
paracrine or autocrine (they affect regions close to where they are produced, rather than the whole body) ￫ in many cells, they regulate synthesis of cAMP
What is the most common way of analyzing and thereby regulating the levels of fat-soluble vitamins in an individual?
partition coefficients
Which tRNA contains the growing polypeptide chain?
peptidyl-tRNA (*P site*)
*Chymotrypsin* cleaves at the carboxyl end of which amino acids?
phenylalanine, tryptophan, and tyrosine
Give a famous example of an esterification reaction in the body.
phosphorylation of glucose in glycolysis
In *column chromatography*, the stationary phase is ____________.
polar (usually silica or alumina beads)
Even though triacylglycerols have oxygen, they are considered nonpolar overall. Why?
polar hydroxyl groups on glycerol are bonded to polar carboxylates of fatty acids, decreasing their polarity
What is the preferred gel for *protein* electrophoresis?
polyacrylamide
What type of cistrons do prokaryotes have?
polycistronic Explanation: mRNA is usually generated from an *operon* as a polycistronic transcript ￫ one that has several *open reading frames* which produce several different proteins) These polypeptides usually have a related function (they often are the subunits composing a final complex protein). Can be started by many different *start codons* and stopped by many different *stop codons*.
Natural rubber
polyterpene isoprene chains b/w 1000 and 5000 units long
When Hill's coefficient > 1, what type of cooperative binding is occurring?
positive (green in picture) Explanation: after one ligand is bound, the affinity of the enzyme for further ligands increases.
Why is a single molecule of hemoglobin very rarely only partially oxygenated?
positive cooperative binding means that after one binds, another will bind with even greater affinity.
vitamin K (phylloquinone, K₁, and menaquinones, K₂)
posttranslational modification of *prothrombin* mnemonic: *K* for *K*lotting addition of Ca²⁺-binding sites on many proteins
What limits the *Bradford protein assay*?
presence of detergent in the sample or excessive buffer
Biological function of waxes in animals
prevent dehydration water-repellant to keep skin & feathers dry lubricant
*Sequencing* is a lab technique that helps determine a protein's ____________ structure.
primary ✴︎uses the DNA that coded for that protein, or can use the protein itself
How is the translation of prokaryotic *rRNA* and *tRNA* different from translation of prokaryotic *mRNA*?
prokaryotic *mRNA* transcription and translation are coupled to each other. They require *no* posttranscriptional modifications. On the other hand, prokaryotic *rRNA* and *tRNA* are transcribed into units considerably *longer* than mature functional ones and hence these precursors *are* subjected to modifications
What is the purpose of *restriction enzymes* in their native state in bacteria?
protect bacteria from infection by DNA viruses
Major and minor grooves of DNA often are sites of...
protein binding
What are oncogenes called before they are mutated?
proto-oncogenes
When pH < pI, the protein is...
protonated, creating a net + charge
Vitamin B₆
pyridoxal phosphate
What molecules splices out introns in rRNA?
rRNA splices out its *own* introns!
Where does α-amylase cleave the polymer?
randomly along the chain, yielding shorters polysaccharide chains, maltose, and glucose
In what type of cells is *telomerase* more highly expressed?
rapidly dividing cells
What happens in general *tautomerization*?
rearrangement of bonds, usually by moving a H and forming a double bond
In redox reactions, the electron *donor* is called a ___________.
reductant (reducing agent) Explanation: it *reduces* the other molecule and it itself is oxidized
What type of enzymes often exhibit cooperative kinetics?
regulatory (*irreversible*/*rate-limiting*) enzymes in pathways Ex: PFK-1 in glycolysis
What aspect of the *polymerase chain reaction* process allows the Taq polymerase enzyme to act specifically and actually replace *helicase*?
repeated heating and cooling cycles
What is the role of eukaryotic *DNA polymerase ɣ*?
replicates mitochondrial DNA *5' ⟶ 3'*
Vitamin B₂
riboflavin
Where does translation occur?
ribosomes in the cytoplasm
What characteristics are found in the products usually formed from *lyase*-catalyzed reactions?
rings or multiple bonds Why? - water is not used, so these help to reform *octets*
What do S values of rRNAs indicate?
roughly indicate molecular size
*Motifs* are repetitive organizations of ___________ structural elements.
secondary
Biological function of waxes in plants
secreted as a surface coating to *prevent excessive evaporation* and *protect* against parasites
What method is used to determine the *primary* structure of proteins?
sequential digestion of the protein with specific cleavage enzymes For *small* proteins ￫ *Edman degradation* For *larger* proteins ￫ digestion with *chymotrypsin*, *trypsin*, and *cyanogen bromide* to create smaller fragments followed by either electrophoresis or Edman degradation ❗️Note, this won't help you determine higher level structure like disulfide links or salt bridges
Phosphorylation in eukaryotes is most commonly seen with which amino acids?
serine, threonine, and tyrosine Think logically about this, they all have -OH functional groups! The bond formed is a phosphoester bond
What do *polymerases* do?
shift nucleotides onto growing strands of DNA or RNA
When is the *5' cap* added?
shortly after transcription initiation, and while elongation is in process So even though it's often classified as a *posttranscriptional modification*, it happens *during* transcription
What changes to a M-M plot occur when *cooperativity* is present?
sigmoidal instead of hyperbolic
Mutations in the *wobble* position tend to be...
silent/degenerate ￫ no effect on expression of the AA and therefore no effect on polypeptide sequence. Illustration: To code for *glycine*, the first two nucleotides on the *codon* must be GG. The third nucleotide could be U, C, A, or G, and the AA composition of the protein would remain the same! (Note, In the example in the picture a single tRNA could bind to the first 3 options, but a different tRNA would be needed for GGG, since I cannot base pair with G)
*Cadherins* often hold (similar/different) types of cells together.
similar Ex: epithelial cells (*E-cadherin*)
Glyceraldehyde
simplest aldose (an aldotriose)
Dihydroxyacetone
simplest ketose (a ketotriose)
What is the head group on *ceramide*?
single hydrogen atom
All chromatography is about the affinity of a substance for the mobile and stationary phases, *except* for which subtype?
size-exclusion
In *chromatography*, components with a high affinity for the stationary phase will migrate at what speed?
slow, if at all Explanation: if it is similar to the surroundings, it will *stick* to them and be slowed. ❗️careful, with *ion-exchange* chromatography, the charged beads attract compounds with *opposite* charges.
What RNA molecules or proteins are found in the spliceosome during *intron excision*?
snRNA, hnRNA, and snRNPs hnRNA is what is being modified (contains introns). ✴︎Note, in the graphic, it says pre-mRNA, but this is a subtype of hnRNA. snRNA and snRNPs form a complex that recognize the 5' and 3' splice sites of the introns, forming a lariat to excise and subsequently degrade the noncoding regions.
What is the equation for *migration velocity* of a molecule undergoing *electrophoresis*?
so more charged molecules will migrate *faster* (directly proportional) and larger molecules will migrate *slower* (v is inversely proportional to f, which is dependent on mass)
Why is the *Lock and Key* hypothesis weakened by the concept of *promiscuous reactivity*?
some enzymes do indeed catalyze multiple reactions, which wouldn't be allowed under this strict model because there would need to be a new site for each different substrate
What must occur in order for cooperativity to be seen?
some substrate must be bound
What is a *palindromic DNA sequence*?
specific double-stranded sequences where the 5' to 3' sequence of one strand is identical to the 5' to 3' sequence of the other strand (in *antiparallel* orientation)
The *concentration* of substrate is determined almost exclusively through what kind of activity analysis?
spectroscopy ① *UV spectroscopy* can be used to analyze proteins without any treatment, but is sensitive to sample contaminants ② BCA assay ③ Lowry reagent assay ④ *Bradford protein assay* (most common, reliable, simple, but less accurate when more than one type of protein is present in solution)
Cholesterol is a biological precursor to what molecules?
steroid hormones bile acids Vitamin D
cholesterol function
steroid that is a component of the phospholipid bilayer ￫ ① mediates *membrane fluidity* ② precursor to steroid hormones, bile acids, vit D
What is glutamic acid's charge in its fully protonated state (low pH)?
still +1 Explanation: 2 carboxyl's (both neutral when protonated) and 1 amino (+ when protonated)
vitamin E (tocopherols)
structure: substituted aromatic ring w/ long isoprenoid side chain antioxidants using aromatic ring
Describe the structure and function of a *ribosome*.
structures where polypeptides (proteins) are built. • Made up of ⅓ protein (allow it to change shape during catalysis) and ⅔ *rRNA* (provide structure/function). • 2 *subunits*, a *large* one and a *small* one, which come together around an mRNA—kind of like the two halves of a hamburger bun coming together around the patty. • The ribosome provides a set of handy slots where tRNAs can find their matching codons on the mRNA template and deliver their amino acids. These slots are called the A, P, and E sites. • Also acts as an enzyme, catalyzing the chemical reaction that links amino acids together to make a chain.
What type of molecules can undergo *mutarotation*?
sugars or any other chiral cyclic hemiacetal
What are the units for *kcat*?
s⁻¹
What type of RNA does RNA polymerase III transcribe?
tRNA some snRNAs 5S rRNA
What factors determine whether an enzyme will be active under different environmental conditions?
temperature, pH, salinity
What are the "ingredients" for DNA *sequencing*? (chain-terminating version a/k/a *Sanger Sequencing*)
template DNA primers appropriate DNA polymerase all 4 deoxyribonucleotide triphosphate dideoxyribonucleotide (in lower concentrations)
____________ are odiferous chemicals that are the metabolic precursors to steroids and other lipid signaling molecules.
terpenes built from *isoprene* (C₅H₈) moieties) carbons grouped in multiples of 5
Disulfide bridges are primarily involved in what type of protein structures?
tertiary
Hydrophobic effects become significant with ____________ protein structure.
tertiary
What is the significance of the polarity of *tubulin*?
the *n*egative end of a microtubule is usually located adjacent to the *n*ucleus (no growth/slow-growing), whereas the *p*ositive end (fast growth) is usually in the *p*eriperhy
What structure is an important component of *keratin*, the fibrous structural protein found in human skin, hair and nails?
the *α-helix*
isoprene
the 5-carbon building block of terpenoids.
Explain the following convention for naming mutations: *E6V*
the 6th amino acid (glutamic acid, E) has been changed to valine (V)
In the repression model, the repressor is *inactive* until...
the corepressor binds
What is *catalytic efficiency*?
the efficiency of an enzyme Mathematically: kcat ----- Km
saponification
the ester hydrolysis (*cleavage*) of triacylglycerols using a strong base
What allows humans to target antibiotics to *bacterial* cells with fewer side effects to humans?
the fact that prokaryotic and eukaryotic ribosomes have slightly different structures
What often serves as the corepressor in repressible systems?
the final product it's negative feedback The system can transcribe until a high level of the product is detected, which can then bind to the repressor and halt transcription
Where does the energy that creates a peptide bond during translation come from?
the high-energy *aminoacyl-tRNA bond* (ester) that was formed during activation.
Why does *Km* stay the same in noncompetitive inhibition?
the inhibitor changes the conformation of the active site so that *no amount of increase in substrate concentration can overcome this*. It can't bind at all!
What is the advantage to *chromatography*?
the isolated proteins are immediately available for identification and quantification
What is the anomeric carbon?
the new chiral center formed in ring closure; it was the carbon containing the carbonyl in the straight-chain form it's the one that forms glycosidic bonds in monosaccharides, it houses the hemiacetal and is the most oxidized carbon in polysaccharides, it houses the acetal C1 in aldoses C2 in hexoses
Distinguish *chromatin* from *nucleosomes*.
the nucleosome is the *subunit* of all chromatin *Nucleosome* ￫ DNA wrapped around one octamer of histones (2 copies of each H2A, H2B, H3, & H4) *Chromatin* ￫ collection of nucleosomes
How is the charge on arginine's side chain distributed?
the positive charge is delocalized over all 3 N atoms
Why is the *Lock and Key* hypothesis weakened by the concept of *reverse catalysis*?
the reaction would not be able to go backward according to this model, but we know that it does! *Le Chatelier's Principle* is a widely accepted theory that claims that most reactions do go backwards
In peptides with *signal sequences*, what must occur in order for the protein to be able to enter the organelle and accomplish its function?
the signal sequence must be *cleaved* Ex: insulin and growth hormone
What is the role of *restriction enzymes/endonucleases* in gel electrophoresis?
they *cleave the DNA* before electrophoresis, which goes on to separate these fragments. *Southern blot* often follows to detect the sequence.
Why are *selectins* unique?
they bind to carbohydrate molecules that project from *other* cell surfaces (note, this type of bond is *weak*)
How do free fatty acids circulate in the blood?
they bond noncovalently to serum albumin
What is a mnemonic to remember the structures for *serine* and *threonine*?
they both have -OH attached to C1 on the side chain however *serine* (*s* comes before *t* in the alphabet) lacks another methyl group so its side chain is just ---CH₂-OH *threonine* (*t* comes after *s* in the alphabet) has an extra methyl group attached to C1 on the side chain, so its side chain is ---CH-CH₃-OH
What happens to the specific rotations of the α and β anomers of D glucose when either of the pure compounds is dissolved in water?
they both reach the same value over time! α starts out with [α] = 112° and ↓ to 52.5° β starts out with [α] = 18.7° and ↑ to 52.5° Explanation: it's no longer a pure compound! Cyclic hemiacetal forms of sugars are in equilibrium with the straight-chain ("linear") form. That means that even if you start with a 100% pure sample of either the α or β anomer, once it has been dissolved in water it can equilibrate, via the straight-chain form, to the other anomer. [If A is in equilibrium with B, and B is in equilibrium with C, then A is in equilibrium with C. That's the Zeroth Law of Thermodynamics]. The 36:64 ratio of alpha (α) to beta (β) represents the distribution of isomers when D-glucose is in equilibrium in water at 25° C https://ibb.co/nrTjkf
How do prostaglandins contribute to pain sensation and inflammation?
they regulate the synthesis of *cAMP*, which is involved in many pathways, including those in pain and inflammation
Describe the structure and function of a *tRNA* molecule.
they're like molecular "bridges" that connect mRNA codons to the amino amino acids they encode. One end of each tRNA has a sequence of three nucleotides called an *anticodon*, which can bind to specific mRNA codons. The other end of the tRNA carries the *amino acid* product specified by the codons (technically, when AA is bound it's called an *amino-acyl tRNA*) It is housed within a *ribosome*, which has handy "slots" (P, A, & E sites) for the tRNA to find their matching *codons* on the mRNA template and then deliver the appropriate AA on the other side.
Vitamin B₁
thiamine
What is the significance of the positive and negative sides of *actin*?
this polarity allows motor proteins to travel *unidirectionally* along an actin filament, like a one-way street
For what type of molecules is *facilitated diffusion* used for?
those that are *impermeable* to the membrane (large, polar, or charged)
Which two amino acids have chiral carbons in their side chains in addition to the chiral α-carbon?
threonine and isoleucine ✴︎Just as only 1 configuration is normally seen at the α-carbon, only 1 configuration is seen in the side chain chiral carbon!
Ultraviolet light induces the formation of dimers between adjacent ____________ residues in DNA.
thymine
When is *PAGE* useful?
to compare molecular size or charge of proteins *known to be similar in size* from other analytic methods.
What is the goal of *DNA cloning*?
to convert and amplify a heterogenous mixture of DNA into a large *homogenous* mixture for other applications
Which enzyme works ahead of the other and why? - helicase - topoisomerase
topoisomerase, to introduce negative supercoils to relieve the torsional strain
*Kinases* fall under what class of enzymes?
transferases Explanation: transfer a *phosphate group* to another molecule
Hemoglobin is most often referred to as what kind of protein?
transport protein *NOT* an enzyme
Collagen has a characteristic _____-helical fiber.
tri
Which terpenes can be converted to cholesterol and various steroids?
triterpenes
True or False: *SDS-PAGE* results in denaturation of the protein.
true Explanation: SDS is a solute
True or False: *DNA ligase* requires an input of energy to seal breaks in DNA.
true https://ibb.co/ibf0m9 Details: specifically it is *adenylated* (complexed with AMP), but different input energy source in prokaryotes and eukaryotes • in *prokaryotes* ￫ uses *NAD*: NAD ⥤ NMD + *AMP* in *eukaryotes* ￫ uses *ATP*: ATP ⥤ PPᵢ + *AMP*
Oxidation of triacylgerols yields _________ the amount of energy per gram as carbohydrates.
twice
What happens to *Vmax* during competitive inhibition? (show on a Lineweaver Burk plot)
unchanged
What type of ion channel is active at all times?
ungated channels are always open
In the induction model, the repressor is *active*...
unless an inducer is present
The phospholipids in the cell membrane have __________ fatty acid tails.
unsaturated Explanation: makes it fluid
vitamin A (carotene)
unsaturated hydrocarbon w/ functions in vision, growth, immunity as retinal ￫ vision (senses light) as retinoic acid (oxidized from retinol) ￫ regulates gene expression during epithelial development
Nucleotides that come before the initiation site are given negative numbers and said to be ____________.
upstream
What biological role do *restriction enzymes* have?
used by the cell to cut DNA *at specific sites* during DNA repair, creating what is known as "sticky ends."
Is mutation to one allele in tumor suppressor genes enough to cause loss of function?
usually *not*, because even one copy of the normal protein can function to inhibit tumor formation. *multiple mutations are required*, in contrast with oncogenes, which can promote cancer with just 1 affected allele
What are some possible names for *transferases*?
usually more straightforward: trans[functional groups]ases or [functional group]transferase exceptions: *kinases* (phosphotransferases) and *polymerases* (nucleotidyltransferases)
Typically, are oncogenes considered dominant or recessive?
usually one 1 copy needed to promote tumor growth, so *dominant*
Why is the *Bradford protein assay* less accurate when more than one protein is present?
variable binding of the Coomassie dye with different amino acids
___________ are *esters* of long-chain fatty acids with long-chain alcohols.
waxes
When will changes in [S] greatly affect the reaction rate?
when [S] is less than Km
A high catalytic efficiency occurs when the value of kcat is ________ and the value of Km is ________
when kcat is *high* and Km is *low*
When is the *Bradford protein assay* most accurate?
when only *one* type of protein is present in solution
When may disaccharides be reducing sugars?
when only one of their two anomeric carbons is involved in the glycosidic bond, meaning that they can convert to an open-chain form with an aldehyde group ex: lactose, maltose
When does a protein stop moving in *isoelectric focusing*?
when pH = pI Explanation: the protein is now neutral, so no motivation to migrate towards either terminal
When is Southern blot useful?
when searching for a particular DNA sequence because it separates DNA fragments by length and then probes for a sequence of interest
What is *supercoiling*?
wrapping of DNA on itself picture an old-fashion telephone cord
What happens to a Lineweaver Burk plot during *competitive inhibition*?
x-intercept closer to origin slope increased Vmax same
Is lactose a reducing sugar?
yes
Is maltose a reducing sugar?
yes
Can competitive inhibitors be overcome?
yes ⟶ increase amount of substrate!
List which DNA polymerases have *low* processivity.
α
Which subunit of the *G protein* actually affects the activity of *adenylate cyclase*?
α Explanation: If αs ￫ stimulates it If αᵢ ￫ inhibits it
Maltose
α-1,4 linkage b/w 2 glucose
Which eukaryotic DNA polymerases contribute most to DNA repair?
β and ε
Cellobiose
β-1,4 linkage b/w 2 glucose molecules
What enzyme cleaves amylose to yield maltose exclusively? At what end of the polymer does it cleave?
β-amylase nonreducing end
How is *amylose* broken down and used for energy by humans?
β-amylase cleaves at the nonreducing end (end w/ acetal), and α-amylase cleaves randomly to yield shorter polysaccharide chains, maltose, and glucose
Which polymerases in eukaryotes have the ability to proofread?
δ and possibly ε, but definitely not α
List which eukaryotic DNA polymerases have *high* processivity.
δ and ε
Map out how an enhancer functions and its component parts.
• *Enhancer* = group of several *response elements*. It is a sequence of DNA (so on the strand) that binds only to specific *transcription factors* which in turn serve as receptors for ligands like *signal molecules* which carry a message. Components: - *Signal molecules* (like cortisol, cAMP, and estrogen) bind to specific receptors on the enhancer. - These receptors are *transcription factors* that use their *DNA binding domain* to attach to a particular sequence in the DNA. - This sequence is called a *response element*. Once all components are bound together, result is increased expression of the gene
What is the difference between *transgenic* and *knockout* mice?
• *transgenic* ￫ cloned gene introduced into germ line (fertilized ova) or embryonic stem cells to look at the effects of that gene. - *Gene integrates randomly*. - Best suited for studying the effects of *dominant alleles*. • *knockout* ￫ gene of interest has been intentionally *removed* or mutated - specific *homologous* recombination (nucleotide sequences are exchanged between two similar or identical molecules of DNA; actual targeted event takes place in only a small % of cells)
What are the *industrial* applications of DNA technology?
• GMO foods (agriculture) ￫ added nutrients, delay ripening, pest resistance; helps with the economy • environmental testing for risk assessment and *cleanup* procedures
What are the differences between *DNA replication* and *transcription*?
• NTPs instead of dNTPs (no deoxy-) • *No* primer • Adds *Uracil* (U) instead of thymine (T) • *RNA polymerase*
What are the *medical* applications of DNA technology?
• human gene therapy • personalized chemotherapy (genotyping tumor cells) • pharmaceuticals: A. recombinant medicines (growing in E.coli versus extracting from humans, ex: insulin, HGH) B. vaccines ￫ now by just recreating outer shell of virus, can inject that instead of a weak virus. - cost effective, and less risk of actually contracting the disease
What are the advantages to using stem cell lines to produce transgenic mice, versus germ lines?
• the cloned genes can be introduced *in cultures* • one can *select* for cells with the transgene successfully inserted via *mating*￫ chimera mice created, then bred to create heterozygotes; heterozygotes then bred to create homozygous transgenic mice.
What are the stabilizing bonds in *tertiary structure*? *Quaternary structure*?
• van der Waals forces • hydrogen bonds • ionic bonds • covalent bonds *same for 4° structure*
What happens during *base-excision repair*?
① *Glycosylase* enzyme recognizes and removes affected base, leaving behind an *apurininc/apyrimidinic (AP)* site, a/k/a *abasic* site. ② *AP site* is recognized by an *AP endonuclease* that removes the damaged sequence from the DNA. ③ *DNA polymerase* fills in the gap with the correct nucleotide ④ *DNA ligase* seals the strand ⑤ DNA methylase then rapidly methylates the daughter strand
What happens during *nucleotide excision repair*?
① *Helicase* cranks open the DNA to form a bubble, and several *excision endonucleases* chop out the damaged nucleotides/s along with a surrounding patch of DNA. ③ *DNA polymerase* replaces the missing DNA ④ *DNA ligase* seals the gap in the backbone of the strand
What are the 4 criteria for aromaticity?
① *cyclic* ② *planar* ③ *conjugated* (alternating single & multiple bonds or lone pairs, creating at least one unhybridized p-orbital for each atom in the ring) ④ *Hückel's rule*: has 4n + 2 (n is any integer) π electrons
Show the steps of *PCR*.
① *denaturation* (96°C) ￫ double helix ⥤ two single strand templates ② *annealing* ￫ *hybridization* of primers to each single strand template (55°C) ③ *elongation* ￫ Taq pol synthesizes a new DNA strand complementary to the DNA template strand by adding free dNTPs from the reaction mixture in the *5'-to-3'* direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxy group at the end of the nascent (elongating) DNA strand. (optimal temp for *Taq* is 72°C) ④ Repeat until desired amount of copies ⑤ Cool reaction chamber indefinitely (allows for short-term storage of PCR products) Optional: if you want to check that the PCR generated the target region, use *agarose/DNA* gel electrophoresis to separate by *size* and the run a *DNA ladder* (has DNA fragments of known molecular weights) on the gel for comparison
What happens during *mismatch repair*?
① A protein complex recognizes and binds to the mispaired base. ② A second complex cuts the DNA near the mismatch, and more enzymes chop out the incorrect nucleotide and a surrounding patch of DNA. ③ A DNA polymerase then replaces the missing section with correct nucleotides ④ DNA ligase seals the gap
List the general steps of isolating proteins and other biomolecules from body tissues or cell cultures.
① Cell lysis & *homogenization* (crushing, grinding, blending into evenly mixed solution) ⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎ ② *Centrifugation* is the initial isolation step ⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎⬇︎ ③ Other isolation techniques, such as *electrophoresis* and *chromatography*
What are the steps to creating a *transgenic mouse* using fertilized ovum? (Note, there are several methods other than this one).
① Cloned gene microinjected into nucleus of a newly fertilized ovum ② ovum implanted into *surrogate* mother. If successful, resulting offspring will contain the gene in *all of their cells*, including germ line cells This means these offspring will pass on the *transgene* to *their* offspring. *Note*, the transgene *coexists* with the animals' own copies of the gene (they have not been deleted). ✴︎ useful for studying dominant gene effects, but not recessive disease b/c can't control # of gene copies that insert into the genome (transgenic mice may each contain a diff # of copies of the transgene!)
What are the two domains that make up a *transcription factor*?
① DNA binding domain ￫ binds to a specific nucleotide sequence in the *promoter* or to a DNA *response element* to help in the recruitment of transcriptional machinery ② Activation domain ￫ allows for binding of several *transcription factors* and other regulatory proteins (e.g. RNA polymerase, histone acetylases)
What happens during *DNA sequencing*?
① DNA sample to be sequenced is combined in a tube with *primer*, *DNA polymerase*, and (DNA nucleotides* (dATP, dTTP, dGTP, and dCTP). The four dye-labeled, chain-terminating dideoxy nucleotides are added as well, but in much smaller amounts than the ordinary nucleotides. ② The mixture is heated to denature the template DNA ③ Then cooled so that the primer can bind to the single-stranded template. ④ Once the primer has bound, the temperature is raised again, allowing DNA polymerase to synthesize new DNA starting from the primer. ⑤ DNA polymerase will continue adding nucleotides to the chain until it happens to add a dideoxynucleotide instead of a normal one. At that point, no further nucleotides can be added, so the strand will end with the dideoxynucleotide. This process is repeated in a number of cycles. By the time the cycling is complete, it's virtually guaranteed that a dideoxynucleotide will have been incorporated at every single position of the target DNA in at least one reaction. That is, the tube will contain fragments of different lengths, ending at each of the nucleotide positions in the original DNA (see figure). The ends of the fragments will be labeled with dyes that indicate their final nucleotide. ⑥ After the reaction is done, the fragments are run through a long, thin tube containing a gel matrix in a process called *capillary gel electrophoresis*. Short fragments move quickly through the pores of the gel, while long fragments move more slowly. ⑦ As each fragment crosses the "finish line" at the end of the tube, it's illuminated by a laser, allowing the attached dye to be detected. ✴︎The smallest fragment (ending just one nucleotide after the primer) crosses the finish line first, followed by the next-smallest fragment (ending two nucleotides after the primer), and so forth. Thus, from the colors of dyes registered one after another on the detector, the sequence of the original piece of DNA can be built up one nucleotide at a time. The data recorded by the detector consist of a series of peaks in fluorescence intensity, as shown in the chromatogram above. *The DNA sequence is read from the peaks in the chromatogram*.
What properties determine lipid properties?
① Degree of *saturation* in fatty acid chains ② *Functional groups* bound to fatty acid chains
Why must testing for reducing sugar occur in *basic* solution?
① First, acidic conditions might hydrolyze any acetals present to hemiacetals, giving a false positive test. ② Secondly, base considerably speeds up the rate of ring-chain tautomerism (i.e. interconversion between the cyclic hemiacetal form and the linear aldehyde form)
Posttranslational modifications include...
① Folding by *chaperones* ② Formation of *quaternary* structure ③ Cleavage of proteins or *signal sequences* ④ *Covalent* addition of other biomolecules (a) *Phosphorylation* (b) *Carboxylation* (c) *Glycosylation* (d) *Prenylation*
What are the *ethical* concerns regarding DNA technology?
① Is it ethical to... - test for life-threatening diseases and potentially terminate a pregnancy? - select or even test for eye or hair color? ② How to choose human test subjects... - Should you communicate results to relatives of someone who has been tested (potential violation of privacy). - Can you carry out potentially risky therapy in someone whose illness makes him/her unable to communicate?
Once an mRNA has left the nucleus, it may or may not be translated many times to make proteins. What are the two key determinants of how much protein is made from an mRNA?
① Lifespan ￫ "how long it floats around in the cytosol" ② How readily the translational machinery (such as the ribosome) can attach to it *small regulatory RNAs* (srRNAs), can control mRNA lifespan and translation. A subtype is miRNAs
How is protein concentration determined using a standard curve with data obtained from the *Bradford protein assay*?
① Samples of known protein concentrations (often BSA [bovine serum albumin] or IgG) are reacted with the Bradford reagent, then absorbance is measured to create a standard curve ② Then, the unknown sample is exposed to the same conditions, and the concentration is determined based on the standard curve (calibration)! ✴︎Curve is plotted as *absorbance* vs. *[standard]*
There are two types of promoter elements in eukaryotes. What are they?
① TATA box (highly *conserved*) ② Initiator (highly *degenerative*, i.e. specified by multiple different codons) - located *downstream* from TATA box - It can signal transcription in the absence of the TATA box - When TATA box is present, it increases efficiency of transcription by working alongside the promoters that bind to RNA polymerase
What are the ways proteins can participate in *biosignaling*?
① as extracellular ligands ② transporters for facilitated diffusion ③ receptor proteins ④ second messengers
What are the two domains found in a *zymogen*?
① catalytic (active) domain ② regulatory domain (prodomain), which must be removed or altered to expose the active site
How does *quaternary structure* make the protein more stable?
① further reduces the surface area of the protein complex (more folding) ② reduces amount of DNA needed to encode the protein complex ③ brings catalytic sites close together, allowing intermediates from one reaction to get shuttled to a second reaction ④ ⁂ most importantly ⟶ induce cooperativity or allosteric effects
What are the subtypes of *tertiary structure*?
① hydrophobic interactions ② acid-base/salt bridges ③ disulfide links
What are the 3 domains found on *enzyme-linked receptors*?
① ligand-binding ② membrane-spanning ③ catalytic (enzymatic)
How can *ligases* be differentiated from *lyases* that are functioning in the reverse direction?
① ligases typically join together *large* molecules, usually of the *same* type ② ligases *require* ATP to join these molecules
What effect would less branching of glycogen have?
① lower solubility ￫ Branches ↓ interactions b/w adjacent chains of glycogen & encourage interaction w/ aqueous env. ② less storage of glucose ￫ without branches, density of glucose cannot be as high, so less can be stored per unit area ③ slower glycogen phosphorylase activity ￫ smaller # of branches means this enzyme has fewer terminal glucose monomers with which to react ④ fewer (if any) α-1,6 linkages
When antibodies bind to their antigen, what are the 3 possible outcomes?
① neutralize ② opsonization ③ agglutination
How is steroid functionality determined? Are they polar or not?
① oxidation status of the 4 rings ② functional group they carry *nonpolar* due to large # of carbons & hydrogens
What possible reactions can monosaccharides undergo (think about their functional groups)?
① oxidation/reduction ② esterification ③ nucleophilic attack (creating *glycosides*)
Out of the 6 classes of enzymes, two classes both *shuttle things around*. What are the names of these classes of enzymes?
① oxidoreductases ② transferases
What functions does *telomeres* have?
① replaces some of the sequence that is lost in each round of replication ② their high GC-content creates strong strand attractions at the end of chromosomes to *prevent unraveling*
3 important signaling lipids
① steroids ② prostaglandins ③ fat-soluble vitamins
What forces allow for the compaction of the enormous of amount of information stored in the minuscule DNA double helix?
① supercoiling ② 1. DNA wound around 2. *histones* ￫ forming 3. *nucleosomes* ￫ many nucleosomes comprise 4. *chromatin* ￫ 5. chromsomes
What is the advantage to *PAGE*?
① the functional native protein can be recovered from the gel after electrophoresis ❗️so long as the gel was not stained! ② more accurately determines the relative *globular* size of proteins compared to SDS-PAGE
What is the drawback of *affinity chromatography*?
① the recovered protein can be bound to the free receptor in the eluent and be difficult to remove Ex: esp. if the eluent was an inhibitor, it would cling strongly ② may also be too attracted to the column in the initial step
What effect might contamination with detergent or SDS have on a activity assay?
① this contamination might yield an artificially increased protein level, leading to lower activity than expected Explanation: because the protein concentration was calculated as higher than its actual value. or ② detergent denatured the enzyme
What are the 3 main types of *ion channels*?
① ungated ("leak") ② voltage-gated ③ ligand-gated
sesquiterpenes
✴︎ "sesqui-" means ½ ✴︎contain 3 isoprene units
Describe the *Bradford protein assay* method of determining concentration.
✴︎ the dye binds to primarily basic (i.e. arginine) and aromatic AA
How are *isomerases* named?
✴︎Notice how they can also be classified as another of the 6 classes of enzymes since they often involve moving electrons or breaking bonds!
sphingomyelins (sphingophospholipids)
￫ considered both sphingolipids and phospholipids ￫ contain a phosphatidylcholine or phosphatidylethanolamine head group (thus, phosphodiester bond) ￫ head groups have *no* net charge ￫ major components in plasma membranes of myelin-producing cells (oligodendrocytes & Schwann cells)
What is the difference between a steroid and a steroid hormone?
￫ steroid is defined by its *structure*: 3 cyclohexane rings and 1 cyclopentane ring ￫ steroid hormone is a molecule within this class that also functions as a hormone, meaning it *travels in the bloodstream*, is active in *low concentrations*, affects gene expression and metabolism
