Molecules to Cells Module 1
Locations of transcription and translation - euks
transcription occurs in the nucleus and translation occurs in the cytoplasm
*inverted*, mirror, and direct repeats
(inverted repeats = palindromes)
Main 2 structural and polarity differences between myoglobin and hemoglobin
*M*yoglobin is *m*onomeric and many of the surface residues interact with water and are polar. Hemoglobin is a *tetramer*. Its subunits have many surface residues that interact with other subunits via hydrophobic bonds and H bonds to form a tetramer.
Which one of the following statements about EUKARYOTIC gene regulation is correct? a) Large polycistronic transcripts are common. b) Most regulation is positive, involving activators rather than repressors. c) Transcription and translation are mechanistically coupled. d) Transcription does not involve promoters.
*b*- true; remember, in eukaryotes, the DNA is highly condensed and you have to open it up before it can be activated. So it's off and needs to be turned on by a protein binding. Proks are on and need a protein (repressor) bound to prevent transcription. a- no; only in proks c- no; impossible because they occur in different parts of the cell for euks; might be true for proks. d- false; promoters involved in transcription for both proks and euks. *If a gene is going to be transcribed, it has to have a promoter.* Doesn't have to have an enhancer or the other sort of 'accessories' but must have a promoter for transcription to begin.
pH = ___ Ka for weak acid = ___. Ka relationship with pH pKa = ___
*pH = -log ([H+])* A 0.1 molar solution of HCl has a pH = -log(0.1) = 1, a 0.01 molar solution has a pH = -log(0.01) = 2, and so forth. *Ka = [H+] [A-] / [HA]* Use this for weak acids as they do not completely dissociate in water. The value of Ka is proportional to the strength of the acid. A larger Ka means more dissociation of the weak acid, thus a stronger acid: higher [H+] and [A-] (bc dissociated more in water), and lower pH. *pKa = -log(Ka)*
The actual cause of cystic fibrosis is a deletion of ___, a critical amino acid in the CFTR gene, in AA position ___ on the gene. What happens to the peptide chain as a result?
-Phenylalanine, position 508 (F508del) -once it gets to the ER, it is degraded due to this mutation. thus, the CFTR protein doesn't make it up the the cell membrane where it normally functions. Doesn't let chloride out so you get thick mucous.
Osteogenesis Imperfecta -what it is, briefly -problem is with Type ___ Collagen -caused by defect in a gene called ___, which makes ___ -why are bones most affected?
-brittle bone disease -Type I Collagen -Most often caused by mutations in the *COL1A1* gene encoding *collagen I* (*mnem:* If it's making *collagen 1*, your bones are *A1*) -The dominance of type I collagen in bone explains why bones are predominately affected
X-linked inheritance: dominant -characteristics -offspring when father affected, mother unaffected -offspring when mother affected, father unaffected
-see picture -all daughters affected, all sons normal -50% chance of passing the mutation to daughters or sons, either could be affected
X-linked Inheritance: recessive -characteristics -offspring when father affected, mother unaffected -offspring when mother carrier, father unaffected (mothers not affected because they have that other big X chromosome to even out)
-see picture -all daughters will be carriers, all sons will be normal -50% chance of passing the mutation to daughters or sons, only boys affected
Ion exchange chromatography -separates proteins based on ___ -what kind would you use to retain positively charged proteins so that only negatively charged proteins come out. -vice-versa
-separates proteins based on their net charges -cation exchange column (has - charge that binds + charged proteins) -anion exchange column (has + charged column that binds to negative charges) *mnem*: can remember them based on what they bind -- e.g., cation exchange binds positive charges.
2 ways to terminate transcription in prokaryotes: -Rho-independent transcription termination -Rho-dependent termination
-there must be a G-C rich *palindrome* in the mRNA strand, and since these palindromic sequences are complimentary, a hairpin loop forms.) This loop is followed by multiple *uracils* --> Causes RNA Pol termination and complex dissociates -termination of transcription by an interaction between RNA polymerase and the rho protein (Rho helicase, see pic) at a run of G nucleotides on the DNA template
Hemoglobin: -2 main functions
-transports oxygen, CO2, and protons; 2 main functions: 1. Transport of O2 from lungs to tissues 2. Transport of CO2 and protons from tissue to lungs for subsequent release
Overview of transcription, including: -enzyme -template vs coding/non-template strand (_' -> _' for each and which one RNA is actually made on) -Direction of transcription
.
Any specific Type 2 Restriction Endonuclease can produce either sticky ends or blunt ends when it cuts up DNA. Define each.
1) *Sticky Ends* --PREFERRED METHOD: -Type 2 Restriction Endonuclease cuts at different places on the two strands (e.g., image, cuts between G and A on each strand), leaving one strand overhanging the other to form a short (~ 4 nt) single-stranded segment. -This overhang will easily attach to a complementary overhang/sticky end, and are thus considered "sticky". -These overhangs can still attach to each other, even if they came from different parent DNA molecules, and the enzyme *ligase* will then covalently link the strands. *Sticky ends therefore facilitate the ligation of diverse segments of DNA, and allow the formation of novel DNA constructs.* 2) *Blunt Ends* -- LESS PREFERRED METHOD: -Other restriction endonucleases produce *blunt ends* in which there are no unpaired bases or overhangs in the end of the fragments. These pieces of DNA can not anneal to each other and hence are more difficult to clone.
Arrangement of collagen fibers in: 1) tendon 2) skin 3) (cartilage) 4) cornea
1) Parallel bundles 2) layered sheets of fibrils 3) (no distinct arrangement) 4) flat sheets stacked crossways to minimize light scattering
Mitochondrial diseases (brief descriptions): 1. POLG mutations (2. Leber's hereditary optic neuropathy 3. MELAS Syndrome 4. Kearns-Sayre syndrome)
1. DNA polymerase gamma (POLG) copies mtDNA. (2. Caused by three point mutations in complex 1 (NADH dehydrogenase) of the electron transport chain. Leads to degeneration of retinal ganglion cells causing sudden loss of vision. 3. Stands for Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes. It is also linked to mutations in complex 1, as well as genes linked to the formation of tRNAs. Leads to muscle weakness, loss of muscle control, seizures, and vomiting. 4. Caused by nonspecific mtDNA deletions. Leads to ptosis, limited eye movement, and atrioventricular block later in life.)
Briefly define these genetic disorders...mutation in: 1) Single gene (Mendelian or monogenic) 2) Multifactorial (complex or polygenic) 3) Chromosomal 4) Mitochondrial (he breezed thru this stuff so make lower priority)
1. This type is caused by changes or mutations that occur in the DNA sequence of one gene. 2. This type is caused by a combination of environmental factors and mutations in multiple genes. For example, different genes that influence breast cancer susceptibility have been found on chromosomes 6, 11, etc. 3. Abnormalities in chromosome structure such as missing or extra copies or gross breaks and rejoinings (translocations) can result in disease. e.g., Down Syndrome 4. Encompasses dozens of diseases caused by mutations that impact mitochondrial function. Includes *POLG mutations, Leber's hereditary optic neuropathy, MELAS Syndrome,* and *Kearns-Sayre syndrome *.
*d*(ACGTCT....) *p*(ACGUCU....)
5' --> 3' "d _x_" means deoxy -- often left off but DNA will be deoxy obviously Small p means that it has a phosphate on 5' C -- just know what's different about RNA
The sequence of the DNA strand used as a template for transcription is GATCTAC. What is the sequence of the RNA product?
5'-GUAGAUC-3' Remember, the template strand is 3' to 5' to make RNA that is 5' to 3'. But since not indicated, the template strand sequence is written 5' to 3', so start on 3' end. So for template strand, go *3' to 5'* when you find complementary pairs and then replace T with U.
Heterogeneous Nuclear RNA (hnRNA)
= The primary transcript (unprocessed RNA that comes right out of transcription) Before 5' cap, poly A tail, or splicing (so it has all the exons in it)
The binding of CRP (cAMP receptor protein) of E. coli to DNA in the lac operon: a) assists RNA polymerase binding to the lac promoter. b) Is inhibited by high level of cAMP. c) Occurs in the lac repressor region. d) Occurs only when glucose is present in the growth medium.
A -- true; that's its main function B is wrong because it needs high levels of cAMP to be able to bind (and low glucose leads to increased cAMP)
Role of Glycine in collagen stability
Glycine is regularly spaced at every third residue, smallest amino acid (having only a H atom as a side chain), allows the 3 helical α chains to pack tightly. In picture, if you look down the middle, you can see that area surrounded by glycines. this stabalizes collagen
What AA is important because its pKa is close to neutral
Histidine; histidine makes proteins more sensitive to pH changes.
Progeria is caused by mutation in what gene? Why is this a genetic but not hereditary condition?
LMNA gene Caused by a mutation in that gene, so it's genetic. But it was not congenital. *Not all genetic disorders
Interpreting RFLP results for paternity test, or for criminal forensics (DNA match test) -- with example
Let's use RFLP technology to determine if Jack is the father of Jill's child named Payle. In this scenario, DNA was extracted from white blood cells from all three individuals and subjected to RFLP analysis. The results are shown in the IMAGE. It seems that Jack could be the father, but to make sure, would want to look at a few other loci too.
beta pleated sheet: -R group orientation -stabilized by __ bonds btw...
Polypeptide chain (β-strand) is extended into zig-zag conformation Adjacent R groups protrude in opposite directions Stabilized by H-bonds between chains (know that H-bonds can occur btw AAs that are far away, unlike alpha helix where they need to be 4 AAs away) Chains arranged side-by-side to form a series of pleats Adjacent polypeptide chains can run parallel or anti-parallel When two or more sheets are layered closely, the R groups must be relatively small, e.g., β-keratin in silk and spider webs have high content of Gly and Ala
Which statement(s) is/are FALSE? a) Phosphorylation of DNA-binding proteins may alter the ability of these proteins to act as transcription factors. b) Tissue specific gene expression does not play a role in the developing embryo. c) Translational repressors bind to the 3' untranslated region of the mRNA and in doing so, may inhibit or slow translation.
a) true -- changes their shapes and function *b*) false -- have to have this c) true
Enhancers are only found in ___.
eukaryotes there are no enhancers for proks. can enhance or silence if a silencer protein binds. they can be anywhere on the gene. In proks, the similar feature would be the activator binding site, but those are very strongly fixed in place (like the -10 and -35)
operon
group of genes with same promoter; have common function
What enzyme unzips DNA/creates replication fork? What enzyme keeps them apart?
helicase single-stranded binding protein (SSB)
Consanguinity
kinship, blood relationship increased risk for auto-recessive DOs
leading vs lagging strand
lead- towards fork, lagging- away from fork Since DNA is antiparallel and DNA is synthesized in the 5' to 3' direction: leading strand (made on 3' to 5' strand) is synthesized continuously lagging strand (made on 5' to 3' strand) is synthesized discontinously - Okazaki fragments
3 types of RNA and their functions Which two are non-coding?
messenger RNA is the only RNA that encodes the polypeptide
What happens to DNA when it's methylated? What happens to histones and DNA when histones are: -acetylated -deacetylated
methylation condenses chromatin; thus, RNA Pol cannot access the DNA to transcribe it acetylation of histones (HAT) reduces histone binding to DNA & leads to transcription. Deacetylation (HDAC) restores chromatin and silences gene transcription.
Concept of multifactoral inheritance
multiple factors affect inheritance.
"Satellite cells" = ___ stem cells Often a point blank question on STEP 1
muscle stem cells i.e., stem cells in mm. are called satellite cells
Explain the oxygen binding curve of myoglobin in lungs, working muscles, and tissue. (think about what myoglobin does)
myoglobin stores oxygen and releases it when mm. need it. So working muscles have low oxygen saturation in myoglobin because myoglobin has released a lot of oxygen. 5-10 mmHg O2 is where oxygen is readily released. In the lungs, myoglobin is the most saturated. (look at the blue line from right to left in the graph)
The ___ is the basic structural unit of chromatin.
nucleosome
Glycerophospholipids are amphipatic containing a polar end (phosphate and alcohol) and a non polar end (fatty acids) The charges on the polar end are on what 2 molecules?
on the phosphate group (negative charges) on the alcohol esterified to the phosphate
p53 induces cell cycle arrest by activating the gene to make p__ (___-___ inhibitor)
p21 (cyclin-dependent inhibitor)
Pka, Pkb, and isolectic pH (pI) on a titration curve
pI is when the total charge on the AA is zero
Recognize cholesterol and identify the polar head group
polar head group is -OH
Most and least abundant type of RNA in the cell
rRNA (mRNA is least abundant)
The nucle*olus* is the site of ___ and ___ production
ribosome and rRNA production
Multiple snRNPs assemble into a RNA/protein complex called the what?
splicesome - RNA/protein complex almost as large as a ribosome Machinery consists of five 100 -300 base snRNAs (U1, U2, U4, U5, U6) and their interacting proteins. SnRNPs are responsible for 3 primary functions: Recognize sites (2 splice sites and branch point site) Bring these sites together Catalyze cleavage reactions that involve the sites
RNA primer
starting point for DNA synthesis Provides a free *3'-OH* that creates a phosphodiester bond with a phosphate
** Changes in Hgb's shape when O2 binds (T -> R state) #1 What happens to the His HC3 residue?
they move to the center of the molecule
Portal vein, Hepatic artery, bile duct on US
vein over artery
Trinucleotide repeats and genetic test (<-- easy) Main example and what 3 letters are repeated?
we all have normal trinucleotide repeats at the end of our DNA, but certain conditions arise when these repeats are expanded (multiplied, A LOT). They require their own genetic test, just called trinucleotide repeat test. Main example is Fragile X syndome! *CGG* repeated
Exons
where genetic info is stored. 85% of disease-causing genetic mutations are in the exons
Microarray / Comparative Genomic Hybridization (CGH) test -What specifically you would want to use it for -what it is -generally what it detects
*CGH is the recommended First Tier clinical diagnostic test in evaluation of: Non-syndromic dev delay/Intellectual Disability, Autism and/or Multiple Congenital Anomalies* Commonly used to determine *if portion of chromosome is deleted/duplicated.* This is *the current best and most commonly used* of the three "cytogenetic" tests, which focus on chromosomes themselves because it shows you the most detail about the chromosomes -- tells you exactly how much of the chromosome is missing and where it's missing from. It also has a much higher probability of detecting chromosomal abnormalities compared to the others. IMAGE: Place equal amount of pt and control DNA in wells; if end up being equal, get yellow. If not, get red or green.
Which statement(s) is/are correct? a) Enhancer elements are always upstream of the TATA box. b) Enhancer elements inhibit transcription and never stimulate transcription. c) Enhancer elements are never found in introns. d) DNA-binding trans-activators bind to enhancer elements and facilitate transcription.
*D* -- can facilitate it or weaken it A is false; can be upstream, downstream, or within intron
euchromatin vs heterochromatin -in terms of definitions and appearance
*Euchromatin*: In interphase cells, the euchromatin is decondensed and transcriptionally-active, and is distributed throughout the nucleus. It is *uncoiled* genetic material (lighter stained) that is *trascriptionally active*. *Heterochromatin*: Heterochromatin is highly condensed and not transcribed, and is often associated with the nuclear envelope or periphery of the nucleolus. It is *coiled* genetic material that is *transcriptionally inactive*.
HgA1c
*Glycated hemoglobin* Hgb is gradually glycated over time as it's exposed to sugars in the blood. An excess amount of sugar in the blood --> excess HgA1c. That's why you can measure it to monitor DM.
US relationship between R lobe of liver and R kidney
*L*iver on *L*eft *Right* Kidney on *Right*
What are globins? So --globin (e.g., hemoglobin, myoglobin) means...
"-globin" means the protein: 1) contains heme 2) stores/transports oxygen -(myoglobin: storage, hemoglobin: transport)
2 steps of tRNA attachment to codon by Aminoacyl-tRNA synthetase Step 1: Formation of Aminoacyl adenylate (*AMAD*) (i.e., it's made from ___ + ___)
(Don't need to be able to draw out structures) ATP + Amino Acid --> Aminoacyl adenylate (*AMAD*)
Define/explain *Activators* and *Repressors* in regulated bacterial gene expression, including where they bind and how they affect gene expression.
*Repressors*: -Bind AFTER the promoter, actually binding to the operator (imagine it binding at the beginning of the operator) and block RNA pol from moving along to transcribe the gene. *Activators*: -bind BEFORE the promoter (so they won't get in the way of RNA pol) and actually enhance the ability of RNA Pol to bind to the promoter region. They INCREASE the expression of the gene.
the human genome contains how many base pairs? How many known genes?
3.2 billion 20,000
RNA overall shape (tertiary structure - 2D) but also 3D shape
3D shape can vary a lot. RNA can adopt more 3D structures than DNA.
Cri-du-chat (mnem)
5p- (disorder in which p leg of chromosome 5 deleted) *mnem*: C5 --- like the musical note, imagine a C5 on piano.
Abdominal Aorta vs. Inferior Vena Cava in longitudinal US views
Abdomina Aorta will have vertebral bodies under it (white disc shaped objects)
R
Arginine
N
Asparagine (Asn)
D
Aspartic Acid
A vs B form DNA -which is more prominent -What form do DNA/RNA hybrids take
B-form is the most prominent DNA/RNA hybrids (usually middle steps/structures in processes) are A form.
Deoxycytidine vs Cytidine
C2
Interpreting RFLP results to see if a person is a carrier for an allele -- with cystic fibrosis (CF) as an example
CF is a recessive disorder, so a diseased person must be homozygous for the diseased alleles.
bicarbonate buffer system
CO2 + H2O ↔ H2 CO3 ↔ H+ + HCO3- Bicarbonate (HCO3-) is in equilibrium with carbonic acid and carbonic acid is in equilibrium with CO2 dissolved in the blood so CO2 effectively acts as the weak acid in this system, and HCO3- is its conjugate base. -Blowing off CO2 (hyperventilation) decreases levels of CO2 which causes reaction to shift left consuming H+ and reducing H+ in the blood making pH less acidic
Genetic test to determine if a person is a carrier for a disease (usually, to determine the risk of a person having children with a particular condition)
Carrier testing
What condition is a karyotype particularly good for?
Down Syndrome because you can see the extra chromosome also microcephaly This is one of the three "cytogenetic" tests, which focus on chromosomes themselves.
DNA Slippage (of DNA Polymerase) and nucleotide expansion (Triplet Repeats). What disease is a result of triplet repeats?
Due to hairpin, you end up replicating and re-replicating Clinical significance: Fragile X syndrome caused by triplet repeats. Longer the repeat, worse the disease.
secondary structure
Either an alpha helix or beta pleated sheet.
Analysis of RFLP results with example
Ex: Looking at a particular RFLP in Jack and Jill. Since they are diploids, each have 2 copies of the RFLP. Jack 1: -GAATTC---(8.2 kb)---GCATGCATGCATGCATGCAT---(4.2 kb)---GAATTC- Jill 1: -GAATTC---(8.2 kb)---GCATGCATGCATGCATGCAT---(4.2 kb)---GAATTC- These are identical. But lets look at their other copies: Jack 2: -GAATTC--(1.8 kb)-CCCTTT--(1.2 kb)--GCATGCATGCATGCATGCAT--(1.3 kb)-GAATTC- Jill 2: -GAATTC--(1.8 kb)-GAATTC--(1.2 kb)--GCATGCATGCATGCATGCAT--(1.3 kb)-GAATTC- When we examine their second copies of this RFLP, we see that they are not identical. Jack 2 lacks an EcoR I restriction site that Jill has 1.2 kb upstream of the target sequence So (IMAGE) we can see in results that they have one band in common and one band that does not match the other's in molecular weight
X-linked inheritance (X-linkage)
No male to male transmission Mothers with 1 "abnormal" X have a 50% chance of passing it to next generation It can be dominant or recessive
F
Phenylalanine
If you want to determine the sequence (spelling) of ONE gene, what test would you use? (mnem)
Sanger sequencing *mnem*: Sanger -- Single
Okazaki fragments are on which strand.
Small fragments of DNA produced on the lagging strand during DNA replication, joined later by DNA ligase to form a complete strand.
How does restriction fragment length polymorphism (RFLP) work?
essentially, have restriction enzymes cut up DNA at particular sites. Fragments of different sizes will come out. Then you can put a DNA probe (with radioactive properties) on there that is complementary to a particular sequence of DNA and measure where it binds.
Number of carbons in: -palmitic acid (saturated) -stearic acid (saturated) -palmitoleic acid (unsaturated) -oleic acid (unsaturated)
-16 -18 -16 -18
DNA mismatch repair
-backup system -corrects 99% of errors -looks for nucleotides not correctly matched up
Chromosomal structural abnormality causes: *Inversions* -- what type of inversion...: 1) involve the centromere 2) involve a single chromosome arm; do not involve centromere
1) pericentric inversion 2) paracentric inversion
Chromosomal structural abnormality causes: *Deletions* -- what type of deletion is each?: 1) deletion of any end of chromsome 2) deletion anywhere in chromosome arm (names are actually pretty simple)
1) terminal deletion 2) interstitial deletion
** Changes in Hgb's shape when O2 binds (T -> R state) Overall, what are the 6 events that take place in Hemoglobin after the first oxygen binds? ...that allows O2 to bind cooperatively
1. The binding of O2 pulls the Fe2+ into the porphyrin plane. 2. The connected HisF8 (histidine on F-helix) moves toward the porphyrin. 3. The binding of O2 changes the position of the F-helix, of which the His F8 is a part. 4. The position of the FG corner of the subunit changes, destabilizing the noncovalent interaction with the C-helix of the adjacent subunit at an α1β2 or α2β1 subunit interface. 5. The adjacent globin structure changes to a conformation that allows O2 to bind to its heme with higher affinity. 6. The new conformation allows the His F8 residues to approach their respective porphyrins with less steric repulsion, thus, O2 binds to the empty hemes in the modified conformation with higher affinity.
Trisomy & Tetrasomy
3 copies of a chromosome (e.g., trisomy 21 = down syndrome) tetrasomy is 4
How many oxygen molecules can be transported by one hemoglobin molecule?
4
A karyotype shows a person has 5 X-chromosomes. How many Barr bodies will this individual have?
4!!! Number of Barr Bodies = Number of X chromosomes - 1. (So 5-1 = 4 Barr bodies)
In higher eukaryotes, the primary transcript of rRNA genes is the ___. How is this processed?
45S pre-rRNA The pre-rRNA is processed via a series of cleavages.
Wolf-Hirschhorn syndrome (mnem)
4p- (p leg of chromosome 4 deleted) -- mnem- *W4* so it's caused by chromosomal structural re-arrangement, De novo or inherited Clinical features: 1. Microcephaly 2. "Greek warrior" faces 3. Mental deficiency 4. Cleft lip / palate
Phosphatidylinositol
A glycerol phospholipid with a head group formed from inositol.
Shelterin
A protein complex that binds to telomeres and protects the ends of the DNA from being inadvertently repaired as a double-stranded break in the DNA.
Protein domain
A protein domain is a conserved part of a given protein sequence and (tertiary) structure that can evolve, function, and exist independently of the rest of the protein chain. Each domain forms a compact three-dimensional structure and often can be independently stable and folded.
** Changes in Hgb's shape when O2 binds (T -> R state) #2 Explain the rotation that occurs in the Hgb subunits when O2 binds and how this relates to Hgb's cooperativity for oxygen binding.
A rotation between α/β subunits also accompanies O2 binding: Upon binding one O2 molecule, one pair of α/β subunits rotates with respect to the other by about 15 degrees, altering Hgb's structure and increasing the affinity of the other hemes for oxygen
Sphingomyelin
A sphingophospholipid containing a sphingosine backbone and a *phosphate* head group.
Which of the following is NOT involved in steroid hormone action? a) cell surface receptors b) hormone-receptor complexes (*what is this?*) c) specific DNA sequences (*these are called ___ ___ ___*) d) transcription activation and repression e) zinc fingers
A- not involved; the steroid is in the cell and binds to receptor inside. b- is involved -- this binds to the DNA and the steroid bind to this (hence hormone receptor) c- is involved -- *hormone response elements* d- is involved; usually activation, but can also be repressors e- IS INVOLVED -- these are part of the receptor
Which of the following is true of aminoacyl-tRNA synthetases? (go over each one) a) they "recognize" specific tRNA molecules and specific amino acids. b) In conjunction with another enzyme attach the amino acid to the tRNA. c) Interact directly with free ribosomes. d) Occur in multiple forms for each amino acid.
A- true B- false; the one enzyme does ALL of the steps. C- false D- FALSE!! One enzyme for each AA
Ribosomal RNA: -relationship with ABx -Staph aureus relationship w/ erythromycin
ABx (e.g., erythromycin) target bacterial ribosomes Staphylococcus aureus can become resistant to erythromycin due to *methylation of specific adenosine residues of rRNA* (by the enzyme methylase).
Ligase uses ___ to stitch together 2 pieces of DNA
ATP
What enzymes attach the correct amino acid to its tRNA? To what end of the tRNA is the AA attached?
Aminoacyl-tRNA synthetases this is the first step of translation Attaches to the 3' end of tRNA
Initiation of translation in proks and euks: Bacteria initiate translation with ___. Eukaryotes initiate cytosolic translation with ___. Eukaryotes initiate translation in mitochondria and chloroplasts with ___.
Bacteria initiate translation with N-formylmethionine-tRNA (fMet or tRNAfmet). Eukaryotes initiate cytosolic translation with a specialized initiating tRNA for methionine. (Met-tRNAimet) Eukaryotes initiate translation in mitochondria and chloroplasts with fMet.
autosomal dominant
Both M and F are affected; M may transmit to M; each generation has at least one affected parent; and one mutant allele may produce the disease. *Vertical Transmission* - you see people affected every generation. essentially means it's passed down through each generation (makes sense since it's dominant) aa (i.e., double recessive) *will not pass the trait on*. If carrier, would not be white shape.
Double strand break repair
Broken DNA initiates homologous recombination that results in rejoining of the broken DNA ends.
4 primary patterns of inheritance
But we're skipping Y-linked. Focus on Autosomal dominant, autosomal recessive, and x-linked inheritance
Diagram of how chaperonins work
Chaperonins bind unfolded polypeptides in their molten-globule state & facilitate correct folding Can see that chaperonins provide a safe place for proper protein folding to occur.
Chromatin during mitosis and interphase
Chromatin becomes highly condensed during mitosis to form the compact metaphase chromosomes. During interphase, most of the chromatin decondenses and is distributed throughout the nucleus. But even in interphase, the chromosomes occupy distinct regions and are organized such that transcriptional activity of a gene is correlated with its position. Each chromosome occupies a discrete region of the nucleus, called a chromosome territory.
Cloning of any DNA fragment essentially involves what four steps?
Cloning of any DNA fragment essentially involves four steps: • fragmentation - breaking apart a strand of DNA • ligation - re-arranging and gluing together pieces of DNA in a desired sequence • transformation - inserting the newly formed pieces of DNA into cells • screening/selection - selecting out the cells that were successfully transfected with the new DNA
glycosidic bond Connects ___ to ___
Connect the sugar to the base
Collagen molecules form ___, which makes them form collagen fibers.
Cross-links That's all you need to know
cystic fibrosis (CF) -what inheritance pattern? -affects all the ___ in the body -messes up transport of what ion? -caused by mutation in the ___ gene
Cystic Fibrosis (CF) is an autosomal recessive genetic disorder of the secretory processes of all exocrine glands that affects both mucus secreting and sweat glands throughout the body. The primary physiological defect is disregulation of chloride ion transport. Mutatuion in the *cystic fibrosis transmembrane conductance regulator (CFTR)* gene, which causes the problem with Cl- ion transport.
What process that produces chromosomal structural abnormalities causes the most severe phenotypic effects?
Deletions
Cardiolipin
Diphosphatidylglycerol, a double phospholipid linked by glycerol.
What are hemoglobinopathies? 2 types?
Diseases involving hemoglobin If a mutation causes a problem with hemoglobin that results in a disease, that disease is a hemoglobinopathy. Ways that hemoglobin is impaired: Mutations can cause instability in the hemoglobin structure, increased or decreased oxygen affinity, or an increase in the rate of oxidation of heme ferrous iron Fe2+ to the ferric state Fe3+, referred to methemoglobin, which cannot bind oxygen 2 types: 1. Structural hemoglobin variants (abnormal hemoglobins) 2. Thalassemia syndromes (i.e., less Hb & fewer RBCs)
How do Hgb affinities for O2 differ between adults and fetuses?
Fetal hemoglobin (HbF) has increased oxygen affinity versus adult HbA, which facilitates transfer of oxygen to fetus from the maternal circulation.
Structural difference between *fibril-forming collagen* and *nonfibrilar collagen*
Fibril-forming collagen has long triple helical chains uninterrupted by globular proteins. Nonfibrilar collagen has some globular proteins that interrupt those long stretches of triple helix.
House-keeping/constitutive genes
Genes that are always expressed at a fairly constant level. This can be a high or low level, doesn't matter so long as there's not much variation in the control of expression of that gene. Makes sense because "housekeeping" infers that these genes have important functions and will probably need to be expressed at constant levels. Called constitutive gene expression, as opposed to regulated (or inducible) gene expression, which require other proteins (repressors and activators) that affect whether the gene will be encoded or not.
genetic vs locus heterogeneity and example of each
Genetic Heterogeneity When a gene can have a variety of mutations that all result in the same phenotype. --ex: cleft palate, cleft lip, etc. Different genes in different locations produce same phenotype. --ex: Spinocerebellar Ataxi
Q
Glutamine
glycerophospholipids
Glycerophospholipids most often contain a saturated fatty acid at C-1 and an unsaturated fatty acid at C-2 of glycerol have a glycerol backbone bonded by ester linkages to 2 fatty acids & by a phosphodiester linkage to a highly polar alcohol head group (which will be part of the molecule's name). phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, cardiolipin.
Gangliosides
Glycolipids with a head group composed of oligosaccharides with a sialic acid (NANA) molecule attached to galactose.
cerebrosides
Glycosphingolipids with one sugar
Globosides
Glycosphingolipids with two or more sugars connected to sphingosine
Translesion DNA Synthesis
Goes *across* a *lesion* Once it gets to lesion, DNA polymerase is removed and a translesion polymerase just fills in a few nucleotides, and normal replication can resume
basic (+ charged) amino acids
HAL histidine, arginine, lysine
2 main types of hemoglobin and what pairs of 2 different polypeptide/subunits (greek letters) is each is composed of? (mainly know for adult hemoglobin) Adult Fetal
Hemoglobins are tetrameric proteins composed of pairs of two different polypeptides (α,β,γ,δ etc). While similar in overall length, the human α (141 residues) and β (146 residues) polypeptides of hemoglobin A (HbA) are encoded by different genes and have different primary structures
pH calculations for buffer systems
Henderson-Hasselbalch equation
heterochromatin vs. euchromatin in terms of DNAse
Heterochromatin is highly condensed, DNase resistant, and inactive in transcription. Euchromatin is dispersed, DNase digestible, and active in transcription. (DNAse degrades DNA)
-How to determine the DNA sequence from the results of a dideoxy sequencing reaction -This is the DNA sequence of which DNA strand?
If all of the reactions from the four tubes are combined on one gel, the actual DNA sequence in the 5' to 3' direction can be determined by reading the banding pattern from the bottom of the gel up (thus, it determines the sequence of the coding strand). It is important to remember though that this sequence is complementary to the template strand from the beginning.
phosphatidic acid
If the head group substituent "X" is -H, the phospholipid is phosphatidic acid, the parent compound.
DNA is replicated in what stage of the cell cycle?
Interphase
How condensed DNA will be in different steps of the cell cycle and why (namely, during interphase and metaphase)
Loose euchromatin during interphase because DNA replication is occuring and DNA needs to be loosened so that all the proteins can access it. During mitosis, tight heterochromatin is predominant. No transcription occurs during mitosis as a result. At metaphase of mitosis, the DNA has been condensed nearly ten-thousandfold.
The process of inactivating one of a female's X chromosomes into a Barr body is called ___ *No matter what, how many X chromosomes and how many barr bodies will a person have?*
Lyonization (X inactivation) which X chromosome is inactivated is random. *# Barr Bodies = Number of X chromosomes minus one.* e.g., 5 X chromosomes, 4 Barr bodies. Can only have one X chromosome.
K
Lysine
Collagen chemical components: -particularly rich in what 3 (technically 4) AAs (*mnem*)? -AA sequence and triplets
MNEM: *GAP* - *G*lycine, *A*lanine, *P*roline/hydroxyproline -Rich in *glycine* (33-35% a.k.a. 1/3rd!), alanine (11%), proline/hydroxyproline (21-23%) -Amino acid sequence is a repeating tripeptide of Gly-X-Y where every third residue is glycine, about 1/3rd of the X positions are proline, and about 1/3rd Y positions are 4-hydroxyproline. -Typical triplet repeats are Gly-Pro-Y and Gly-X- Hyp, where X and Y are any amino acid
Effect of cholesterol on membrane fluidity
Makes the membrane more rigid
If you're interested in seeing if a chromosome came from mom or dad or if which parent it came from matters, what would be a good genetic test to use?
Methylation test e.g., IMAGE -- these diseases are caused by only having one copy of the chromosome and can tell which parent it came from.
Abdominal Aorta vs. Inferior Vena Cava in transverse US views (which circle is on left vs right, which one is above the other?)
Mickey Mouse! AA - Right (AR) side of image IVC - Left (IV *L*eague) side of image
What attaches to the centromere, allowing the sister chromatids to separate and move to opposite poles.
Microtubules of the mitotic spindle
Exonuclease, DNA Polymerase, and proofreading
Most of the time, Polymerase will put the correct nucleotide. But there is a process called proofreading to correct mismatching. Exonuclease (part of polymerase) will excise the wrong nucleotide and give Polymerase another chance to add the correct nucleotide.
xeroderma pigmentosum (XP)
Mutated single strand nucleotide excision repair gene, which prevents repair of thymidine dimers.; Dry skin w/ melanoma and other cancers. AR; defect in DNA repair enzymes; ↑ risk for UV light cancers
A mutation in the first base (letter) of a codon would produce what type of mutation? Why?
Mutation in the first base of a codon usually produces a conservative substitution. "missense" mutation―new amino acid is similar in chemical structure
incomplete penetrance
Not all individuals with a mutant genotype show the mutant phenotype the female with the arrow CAN still have affected children, just like the male had a normal phenotype but had affected kids.
___ ___ ___ are the only channels for small polar molecules, ions, and macromolecules between the nucleus and cytoplasm (embedded in nuclear envelope)
Nuclear pore complexes
Weird exception -- methionine has only one codon (AUG), but there are two methionine tRNAs. What does each do, briefly?
One is used exclusively for the initiating methionine, the other for internal methionine residues.
Marfan syndrome and Progeria are examples of the ___ effect
Paternal Age Effect also short limbs
which eukaryotic RNA Polymerase makes each of the 3 types of RNA during eukaryotic transcription?
Pol I: synthesizes pre-ribosomal RNA, the precursor for 18S, 5.8S and 28S RNA. Pol II: synthesizes mRNA and some specialized RNAs. Pol III: synthesizes tRNA, 5S RNA and other small specialized RNAs. [eukaryotes have 3 types of RNA polymerase where bacteria just have the one]
The recognizable banding pattern of collagen fibers is a result of
Post-translational processing of collagen at regularly spaced intervals
What type of genetic tests determine the risk of a person developing a certain disease or condition that they don't currently have (e.g., risk for breast or prostate cancer) (name makes sense)
Pre-symptomatic genetic testing
G-rich DNA sequences can fold into ___
Quadruplex structures just memorize it
RNase H
Removes RNA primers from DNA
Chromosomal structural abnormalities: ___ chromosomes arise when telomeres are lost on both ends
Ring chromosomes
Affinity chromatography (makes sense from the name)
Separates proteins based on some molecular attraction between certain proteins and the medium.
What two very general techniques are required to understand the structure and function of a protein?
Separation and purification --- getting the protein by itself so you can study it. Because there are a ton of proteins in a cell.
*NEED TO KNOW*: If there is a Robertsonian translocation involving chromosome 21 of a female, what will 100% happen?
She will have a child with Down syndrome. 100%.
-relationship between FA chain length and solubility -relationship between FA chain length and melting point -relationship between FA # double bonds and melting point
Solubility -decreases as the chain length increases Melting Point -decreases as the chain length *decreases* -decreases as the number of double bonds increases
What are the structures of A, B, and O antigens? *What type of molecule are these?* *Really* what you need to know is that A and B have one extra sugar attached and what those are.
Sphingolipids The A antigen has one more N-acetyl galactosamine than the O antigen. The B antigen has one more galactose than the O antigen.
3 stabilizing factors of DNA
Stacking interactions exist due to pi bonds (from aromaticity); shown in image; very important for stabilizing duplex DNA, as all 3 of these are. Hydrophobic regions are on the inside, phosphates are on outside. Stabilizing bc DNA really wants to keep like in contact with like H-bonding
If a tissue is deprived of oxygen, it will release VEGF (pron. vej f), and ultimately this will cause the tissue to
Start replicating / duplicating so you get more of those tissue/endothelial cells
** Steps of protein import from the cytoplasm into the nucleus Importins dependent on *what molecule*?
Start this video at 0:54: https://www.youtube.com/watch?v=Q7F2vGwfsL8 1) *Importins* (which are receptors, IMPORT proteins INto the nucleus) recognize nuclear localization sequences on specific proteins and bring them in. 2) These importins are bound by *Ran*, a GTP-binding protein that controls the directionality of transport, ensuring that these proteins come into the nucleus. 3) Once the importins transport the protein complex across the membrane, Ran/GTP binds to the importin to move it back across the membrane. 4) Once this reaches the cytoplasm, *Ran GAP* converts the GTP on Ran to GDP, releasing the importin. 5) The Ran/GDP formed in the cytoplasm is then transported back to the nucleus by its own import receptor, where Ran/GTP is regenerated.
How does systemic lupus erythematosus (SLE) (aka Lupus) work -- what gets messed up?
Systemic lupus erythematosus (SLE): autoimmune disease where patient produces antibodies against snRNPs. If snRNPs aren't present, proper splicing of introns cannot occur.
first step of Pol II eukaryotic transcription (making mRNA) what binds first and where does it bind (names go together!)
TATA Binding Protein (TBP) binds to eukaryotic TATA box at -30. Then other proteins come and join to build the RNA Polymerase. Know that eukaryotic transcription does have *elongation factors*, but don't need to know names.
What method determines the AA sequence of a protein? How does it work?
Tandem Mass Spec degrade protein to peptide and then use mass spec to degrade down to smaller peptide fragments that you can then sequence.
Red blood cells carrying CO2 --- where in the body will you find different compounds of the bicarbonate buffer system? What enzyme does this?
The bicarbonate buffer system controls blood acidity; CO2 is a weak acid so don't want it to be present in blood. Convert it to H2CO3 to prevent blood acidity. The CO2 produced diffuses into blood and enters red blood cells There the CO2 is rapidly converted by the enzyme carbonic anhydrase to carbonic acid, which spontaneously dissociates to HCO3- (bicarbonate) and H+ The HCO3- diffuses out of the red blood cell and is carried to the lungs in the plasma and it turns to So HCO3- is its traveling state -- it's in this form in RBCs. When it gets to tissues, converts to O2; when it gets to lungs, converts to CO2.
Cooperativity and DNA formation
The first step is slow, but once there are a few base pairs, it starts zipping up fast.
Major vs Minor Grooves Significance of major groove
The major groove is the important one of the two. Access to DNA is often along major groove, which is "where the recognition occurs"
O2 saturation curve for hemoglobin and cooperativity. How does the affinity for oxygen affect the transport of oxygen?
The oxygen saturation curve is sigmoidal, denoting cooperative binding kinetics - after one oxygen binds, subsequent O2 molecules bind more readily. Since Hgb transports O2, don't want it to like O2 too much or too little. So it changes how much it likes O2 depending on how many oxygens are already bound. See image -- only middle line is the curve. Low affinity state in tissues (so it can get rid of oxygen) High affinity state in lungs (so it can bind oxygen)
Ceramide
The simplest sphingolipid, with a single hydrogen as its head group.
Real-time PCR or quantitative PCR (q-PCR)
There are several variations of the PCR technique. One commonly used and important variation is real-time PCR or quantitative PCR (q-PCR). Real-time PCR can be used to count the amount of DNA, or number of copies of a gene, present in a sample. It is employed to determine the viral load of HIV in AIDS patients, and also in cancer diagnostics to count the number of cancerous cells remaining in a patient undergoing treatment.
Klinefelter syndrome and Clinical Features and prevalence and chromosomal abnormality (*mnem*)
XXY in males (Mnem: *Calvin Klein has XXY*) Variance- can also be XXXY or XXXXY Clinical Features: 1. Tall, thin male with long legs 2. Underdeveloped secondary sex characteristics 3. Small testicles 4. Gynecomastia (= 50%) 5. Infertility 6. Etiology - nondisjunction
How to use Henderson-Hasselbalch equation with bicarbonate buffer solution. ex: a patient has blood pH of 7.03 and the concentration of CO2 is 1.10 mM
[HCO3-] in this patient's blood is 9.4 mM.
When reading gel pattern for DNA sequence, which direction do you read? (If the ddNTPs are in columns)
bottom to top! The top is the lightest stuff, so it traveled furthest.
US relationship between spleen and L kidney
can see that spleen makes that curved shape; above and left (in image) of kidney.
Constitutional vs. Acquired chromosomal abnormalities (mutations)
cancer is an example of acquired constitutional = germline mutation acquired = somatic mutation
the region of a chromosome to which the microtubules of the spindle attach, via the kinetochore, during cell division.
centromere
clamp loader protein
clamps onto DNA and binds sliding camp protein which holds Polymerase on the DNA
De novo mutations and what pattern of inheritance this would probably occur in
common in autosomal dominant inheritance parents and siblings not affected, it just appears randomly, and from that point on, *it is a dominant trait*
How are embryonic stem cells obtained? (very general, like you get them from where and then do what)
get them from embryo (of a woman who does not want to have more kids) and culture them so they can replicate.
- Extra Y chromosomes clinical features (XYY) - Y-linked genes
Extra Y chromosome Subtle features of tall stature, dis-coordination, speech delays, behavior differences -Genes located uniquely on the Y chromosome are said to be Y-linked. -These genes are passed exclusively from father to son. There are very few confirmed Y-linked disorders. -There is one report of male to male only transmission of a form of an eye disorder that has been tentatively assigned Y linkage. -hair growing from the ear lobe, has also been suggested as possibly Y linked
3 stop codons and start codon
UAA, UAG, UGA start codon: AUG; encodes methionine
Post-transcriptional regulation of RNA -- *RNA interference (RNAi)*
(Don't need to memorize all steps on slide, just basic idea of what's on this card) RNA is ss but can take secondary and tertiary structures. So long stretches of complementary base pairs can form a double helix. Then Dicer protein comes in and sees ds and chops it up. Then a single strand of RNA from one of those chopped up double strands is attached to an mRNA and can either: 1) target it for degradation 2) block translation of the mRNA so that little piece of RNA is what causes RNA interference as it interferes with translation occurring.
Which of the following statements about regulation of the lac operon is true? a) Glucose in the growth medium decreases the inducibility by lactose. b) Glucose in the growth medium increases the inducibility by lactose. c) Its expression is regulated mainly at the level of translation. d) The lac operon is fully induced whenever lactose is present. (inducibility = opposite of repressibility)
(inducibility = opposite of repressibility) A -- when glucose is present, the lac operon does not need to be on. D is wrong because the presence of lactose doesn't matter for this; only the level of glucose. So you need to know how much glucose is present to know how the lac operon will be affected.
One way to determine a person's proteome is to use (2D) gel electrophoresis. This separates proteins based on __ and __. Which proteins travel furthest?
*2D gel electrophoresis* a laboratory method that separates proteins according to their isoelectric points and molecular weights (charge AND size); lighter ones travel furthest. Positive stays with positive and vice versa I think.
Which statements are correct? a) Transcription factors share common protein motifs. b) Tissue specific gene expression can be conferred via specific DNA sequences. c) Multiple enhancer and/or silencers often regulate a single gene. d) Hormone response elements are unique for different hormones and do not share consensus elements.
*A*- true; remember the helix-turn-helix, etc -- those are common motifs. *B*- true *C*- true D- FALSE; first part is true about them being unique for different hormones; but you can line up all HREs and you'll see a common core of bases common to each one.
In what order are AAs in a protein written in?
*Amino* terminal end first
2 steps of tRNA attachment to codon by Aminoacyl-tRNA synthetase Step 2: Attachment of Aminoacyl Adenylate to tRNA (2 options: Class I and Class II Aminoacyl-tRNA synthetases)
*Class I aminoacyl-tRNA synthetase*: transfers aminoacyl adenylate to the 2'OH of tRNA and then to the 3'OH of tRNA, yielding aminoacyl-tRNA (the AA on the tRNA). *Class II aminoacyl-tRNA synthetase*: (simpler) just take the aminoacyl adenylate and transfer it to the 3'OH of tRNA, leaving the AA bound to the tRNA (aminoacyl-tRNA).
Many importins and exportins are members of a family of nuclear transport receptors known as ___.
*Karyopherins* Karyopherins are a group of proteins involved in transporting molecules between the cytoplasm and the nucleus of a eukaryotic cell. (The inside of the nucleus is called the karyoplasm/nucleoplasm.)
A gene with 4 exons and 3 introns produces a mature mRNA. How many bands would you see on a Northern blot of mature mRNA?
*One* Mature RNA -- so it's been spliced and joined back together. introns spliced out, leaving you with the exons that combine to make one mRNA. (Prokaryotes with polycistronic DNA could have 4 RNAs/proteins)
Cell-free fetal DNA testing and main limitation
*SCREENING* test ONLY, not diagnostic. You take pregnant woman's blood, 3-5% of which is fetal DNA, and use it to see if you notice any genetic abnormalities about the fetus. Limitations: Large number of false positives and negatives for certain disorders; *SCREENING ONLY!* -- so if you find something abnormal, it cannot be confirmed until the baby is born and you can do further genetic tests, but it's a good first step.
Tandem Repeats and Slipped DNA
*Tandem repeats can lead to "slipped" DNA* Images a, b, and c: a) *Presence of two adjacent tandem repeats can give rise to two isomers of slipped, mispaired DNA. * b) the second copy of the direct repeat in top strand pairs with first copy of repeat on bottom strand. c) pairing of first copy of direct repeat in top strand with second copy of direct repeat in bottom strand. Two single-stranded loops are generated in each isomer. I guess imagine it just slipping and then the pairs don't match up so it forms a loop.
What genetic test determines the sequence/spelling of all the exons (coding parts of DNA), but not introns? (name makes sense)
*WES - Whole Exome Sequencing* -- because you sequence at the whole exome. Exome = all the exons. Limitations: May also identify unexpected information Risk of Cancer Non-paternity Consanguinity Identification of Variants of Uncertain Significance Does not fully cover all regions of DNA (introns) Limited understanding of resulting information Inability to identify trinucleotide repeat disorders, chromosome rearrangements Expensive
All of the following are required for transcription EXCEPT: a. DNA template b. DNA-dependent RNA polymerase c. Primer d. Nucleotides why?
*c. Primer* Primers are needed for DNA replication, but not transcription. For nucleotides, specifically, transcription requires ribonucleotides. (Replication usually uses deoxyribonucleotides I think.)
*Dominant negative* effect p53 mutation
*dominant negative* = production of a nonfunctional protein that prevents the normal protein from functioning Mutant p53 displays a dominant negative effect as it actually prevents regular p53 from binding to DNA. It does this by binding all over normal p53s (oligomerization), making them inactive (image).
If you want to look at multiple genes at one time (particularly, the sequence/spelling of those genes), and especially when those genes are looking towards a specific phenotype, what test would you use?
*next generation sequencing* -- multiple genes these groups of tests are called panels, where the name of the panel is usually the phenotype in question.
At what 2 places on the prokaryotic *promoter* sequence (which contain DNA *consensus sequences*) does the σ subunit of the RNA polymerase bind to start transcription? Where is the TATA box? Where does transcription actually begin? If you see -10 and -35 on an exam question, you should automatically think ...Aha! this is about ___!
- σ subunit of the prokaryotic RNA polymerase binds to *-10* and *-35* of the promoter. - *-10* is the *TATA box*! - negative numbers mean it's upstream from the starting point, but the starting point of transcription isn't at 0. It's at *+1* If you see -10 and -35 on an exam question, you should automatically think -- Aha! This is about transcription in prokaryotes!
cDNA library vs genomic library in terms of what they are and how to make them
-*Genomic library* made by taking all DNA from an organism, cutting it up (restriction endonuclease), combining it into a plasmid using DNA ligase, and transforming that plasmid into the bacteria. -*cDNA library* made by taking the mRNA from an organism, using reverse transcriptase to produce a complementary DNA strand (cDNA), add restriction sites to ends of cDNA to protect it, then use normal method of cutting it up, inserting into plasmid, then transforming into bacteria.
- RNAs are transported to the cytoplasm as ___ ___. - ___ ___ export tRNAs, rRNAs, miRNAs to the cytoplasm. - how is *m*RNA exported from the nucleus to the cytoplasm? Does not require ___ or ___.
-*Ribonucleoproteins* (RNPs). -Karyopherin exportins (remember karyopherins are a group of proteins involved in transporting molecules, mainly proteins and RNAs, between the cytoplasm and the nucleus) export t, r, and miRNAs. -(image) mRNA transport *does not involve karyopherins* and is *independent of Ran*. A distinct transporter complex moves the mRNA through the nuclear pore. *Helicase* on the cytoplasm side releases the mRNA and ensures unidirectional transport.
Myoglobin: -function -location in body -location on myoglobin of polar and nonpolar AAs and what AA (2 of them in myoglobin) is the exception to this? -rich in what secondary structure only when heme is bound
-*stores* oxygen -located in muscles so that it can release oxygen during low oxygen times -Surface has polar residues and interior has mainly non-polar residues. Residues with polar and non-polar regions (Thr,Trp,Tyr) orient their non-polar regions inward. Exception: two His residues that function in oxygen binding in the interior of the molecule -Myoglobin is rich in alpha-helical regions (8 of them), which decrease dramatically when heme is removed, showing that the heme group contributes significantly to overall structure
Prokaryotic RNA Polymerase *Holoenzyme* (remember, bacteria RNA Pol is already assembled and just binds to the DNA) for transcription of prokaryotic DNA: - β and β' proteins/subunits - σ protein/subunit
-*β* and *β' subunits* form *active sites* of transcription / RNA synthesis (see image for further description) *BS* -- beta, synthesis. -*σ subunit* is the part that recognizes and binds to the promoter region of template strand of DNA (at the -10 and -35 subunits of the promoter) (see image for further description). (*mnem*: *Sr.*, sigma recognizes promoter; or sigma symbol looks like it has a little tail for binding)
Structure of Myoglobin: -how many alpha helical regions? -how are they labeled -on which alpha helical regions would you find the proximal histidine and the distal histidine -role of each of the two histidines in myoglobin's structure (mainly distal his)
-8 -alphabetically, A-H -Backwards from what you'd think alphabetically. Proximal His is on F, Distal His is on E. -Both of them help iron bind to oxygen, but *only the proximal his binds with iron*. -Distal Histidine: (see image) -- it's role is to keep those oxygens from interacting with Fe, thus increasing Fe's affinity for external oxygen. It also keeps carbon monoxide from binding to myoglobin. -Proximal his binds to Fe, but its main role involves conformational changes in hemoglobin.
Addition of 5' cap in RNA processing. -What molecule is the 5' cap? -Function of cap -What is unique about the bond that is formed to add the cap?
-A *7-methylguanosine* cap is added to the 5′ end of the pre-mRNA while elongation is still in progress. -The 5′ cap protects the nascent mRNA from degradation and assists in ribosome binding during translation. Involved in translation initiation This is a *5' to 5'* linkage, which is unique because usually you have 5' and 3' attaching. This is why it's protected -- enzymes do not recognize this linkage. DON'T need to memorize steps, like ignore right sided image.
Genetic condition vs Hereditary condition vs Familial condition
-Based on changes in DNA -- If you change DNA and that gives you a condition, it's a genetic condition. -Inherited thru family -Not necessarily genetic, e.g., family members can all be exposed to environmental factor
Types of Hemoglobinopathies: -Unstable hemoglobins -Increased Oxygen affinity -Thalassemias (α vs β)
-Caused by AA substitutions that don't go well --- make AA unstable. Unstable Hbs denature and precipitate causing the formation of Heinz bodies, which damage the erythrocyte membrane -Some mutant Hbs have an increased oxygen affinity. This impedes the regulation of oxygen dissociation by hydrogen ion concentration and tends to stabilize the R form, leading to increased oxygen affinity and decreased release of oxygen to the tissues. -due to reduced synthesis of either the α subunit (α-thalassemias) or β subunit of Hbg (β-thalassemias) of hemoglobin. A wide variety of mutations are responsible, including those that affect transcriptional regulation and mRNA splicing, as well as those that affect protein function (non-sense, frameshift). The result is anemia, which may be severe.
With skewed inactivation / skewed lyonization, the cell will usually turn off which X chromosome? But what event makes cells often turn off the normal X chromosome?
-Cells prefer to inactivate a structurally abnormal X chromosome (i.e., one with a deletion), leaving the cell with the normal X active -But with balanced X-autosome translocations (e.g., a portion of X chromosome on chromosome 13 and a portion of 13 on the X), the cell will usually inactivate the normal X chromosome.
T vs R state of hemoglobin changes in ___ structure of hemoglobin based on ___ which is more compact? why?
-Changes in *quaternary* structure based on *if oxygen is bound* *T state* = "*T*ense" state; *deoxyhemoglobin* is bound --- no O2 bound to Hgb. -hemoglobin is *more compact* in this state because it has more *salt links* between chains and the lack of O2 allows more covalent bonding. The bond lengths get a little shorter when oxygen leaves. *R state* = "*R*elaxed" state; *oxyhemoglobin* is bound -- Hgb is carrying O2 in this state.
chromosome conformation capture (3C) -what it reveals about chromosomes (what it's used to determine) -briefly how it works. -cross-linking
-In living cells, chromosome conformation capture (3C) techniques reveal sites of interactions between chromosomal regions. -Sites of interaction are identified by *cross-linking* DNA sequences that interact with e/o, which are then amplified and identified.
All about Lamins
-Lamins are a class of intermediate filament proteins that associate to form higher order structures. -Two lamins interact to form a dimer: the α-helical regions wind around each other to form a coil. -The lamin dimers associate with each other to form the lamina. -Lamins bind to inner membrane proteins such as emerin and lamin B receptor (LBR). -They are connected to the cytoskeleton by LINC protein complexes. -Lamins also bind to chromatin.
Effect of 2,3-DPG/BPG on Oxygen binding to hemoglobin When is it high and when is it low?
-Produced in red blood cells under conditions of low oxygen such as anemia and high altitude - 2,3-DPG decreases affinity of hemoglobin for oxygen enhancing oxygen unloading Makes sense about low O2 -- like high altitude, don't feel like breaths are as satisfying.
Transcription enzymes for rRNAs, which make up ribosomal subunits: -The *5.8S, 18S, and 28S* rRNAs (subunit components of euks) are transcribed by the enzyme ___, yielding a 45S ribosomal precursor RNA. -The *5S* rRNA is transcribed by the enzyme ___.
-The 5.8S, 18S, and 28S rRNAs are transcribed by RNA polymerase I, yielding a 45S ribosomal precursor RNA. -Transcription of the 5S rRNA is catalyzed by RNA polymerase III.
Nuclear Lamina -def -image (electron microscope) -consists of fibrous proteins called ___
-The nuclear lamina is a fibrous mesh on the inner side of the nuclear membrane that provides structural support. -Consists of fibrous proteins called *lamins*
the nuclear envelope consists of (3)
-Two nuclear membranes -An underlying nuclear lamina -Nuclear pore complexes
Ehlers-Danlos Syndrome Type IV: -what it is, briefly (most severe effect on what part of body?) -problem is with Type ___ Collagen, which is particularly important in ... -caused by deficiency in what enzyme? Why?
-Type IV Ehlers-Danlos syndrome (*vascular* type) is caused by defects in *Type III collagen*, which is particularly important in *skin, arteries,* and *hollow organs.* -The clinical features include marked hyper-extensibility of the skin and joints, poor wound healing, and musculoskeletal deformities. -Type VI Ehlers-Danlos syndrome is caused by a *deficiency of lysyl hydroxylase* Collagen with decreased hydroxylysine content will have *unstable cross-linking of collagen fibrils*.
Pleiotropism -def -disease example
-When a single gene produces multiple different traits (phenotypes) -Kabuki Syndrome -- deformed fingertips, intellectual impairment, etc --- all of these phenotypes are from the mess-up of one gene.
-___ is having more chromosome material than you should. -___ is a subset of that in which the person has entire extra chromosomes (e.g., Down Syndrome; other triploidy or tetraploidy conditions).
-aneuploidy -polyploidy (triploidy and tetraploidy --- entire extra chromosomes). One set of chromosomes has 23 chromosomes. This is called a haploid set. Two sets, or 46 chromosomes, are called a diploid set. Three sets, or 69 chromosomes, are called a *triploid* set. Four sets, or 92 chromosomes, are called a *tetraploid* set.
pseudoautosomal region
-any genes located within them are inherited just like any autosomal genes. -Males have two copies of these genes: one in the pseudoautosomal region of their Y, the other in the corresponding portion of their X chromosome. -*So males can inherit an allele originally present on the X chromosome of their father* -*Females can inherit an allele originally present on the Y chromosome of their father.* -During male gametogenesis, recombination between X and Y chromosomes occurs in a small subtelomeric region of each the X and Y chromosomes, the pseudoautosomal region. These segments are homologous -Recombination in the pseudoautosomal region guarantees disjunction of X-Y chromosome pairs during male gametogenesis
Mosaicism What is it? What is the main sign that a person has it? How would you check to see if your suspicion of mosaicism is correct?
-essentially, it's when not every cell in your body has the same chromosomal makeup. -Main sign -- skin pigmentation; different colors on skin; not uniform. Major clue to someone having mosaicism. -Can compare cells from one pigment area to cells from another. Can't just look for chromosomal abnormalities by drawing blood and using blood cells. If you think they might have mosaicism, take a biopsy from the abnormally pigmented area of skin. Obviously something is causing that.
Proteins that must enter the nucleus have amino acid sequences called ___ ___ ___. These signals are recognized by ___ ___ receptors.
-have *nuclear localization signals* (NLSs) -*nuclear transport receptors*/ *importins* recognize those / bring them into nucleus -when the same sequence is attached to a different protein, that protein will go into the nucleus
Transcription basics: -RNA polymerase pushes away which DNA strand and makes RNA based on which strand. -Direction that the RNA strand is synthesized; this means that RNA polymerase travels what direction along the template strand of DNA?
-logical -- uses *template strand* (*5' --> 3'*) of DNA to make RNA; pushes away *non-template / coding strand* (*3' --> 5'*). So RNA is made based on template strand, NOT coding strand. Coding strand tells you what the mRNA sequence will be. -*5' --> 3'* so 5' end is made first -so RNA pol travels *3' --> 5'* along the template strand (since it's anti-parallel)
What 3 post-transcriptional processing events must occur to turn RNA into a mature molecule? Primary transcript/Heterogeneous Nuclear RNA?
1) Addition of 5' cap 2) Addition of poly-A tail 3) splicing out introns and connecting exons together Immature RNA is called the *primary transcript* (bc it's what comes right out of transcription) and is only found in the nucleus. By the time it reaches cytoplasm, should be mature.
Size-exclusion chromatography and good analogy for how it works (parents and kids at store) Separates proteins based on ___.
-separates proteins based on size -big proteins=parents, little proteins=kids; parents just want to get through store, kids want to see everything so it takes them longer to get through. -has pores in it that only smaller proteins can fit through; passing through them slows smaller proteins down. Big proteins pass right along and come out first. So which will come out first? Whichever protein is LARGER.
Sickle Cell Anemia: - __ gene disease - Caused by a *___* mutation (*__*aa*__* *--> __*aa*__*) *____ subunit of hemoglobin*. -What property of the new AA is important for what happens to RBCs? (helps with remembering this as a story) - This mutation in sickle cell hemoglobin (termed Hb-S) reduces the solubility of ___hemoglobin, making it precipitate out in RBCs, leading to the sickle shape. -Sickle cells form *what* in blood? -only expressed in individuals with what genotype? -what happens with people heterozygous for sickle cell; relationship w/ malaria.
-single gene disease (only involves problem with one gene) -Glutamate is negatively charged, so you're making the molecule polar -- making it stickier. -*missense* mutation of *V --> E* on the *β-subunit of hemoglobin* -*deoxyhemoglobin* precipitates --> RBC becomes sickle-shaped. -in blood, sickle cells stick to e/o and form pretty long fibers/strands. (image) -homozygous for HbS -50/50 HbA/HbS; experience sxs only when there's a severe lack of oxygen; resistant to malaria.
Effect on transcription when mutation/etc is located: -on 5' end in the promoter region -5' cap -splice donor (5' end), splice acceptor (3' splice site), branching site -in the coding region/sequence
-transcription blocked totally -message gets degraded; can't translate -mess up splicing, get different protein or no protein. Will keep introns or get exons in incorrect sequence. -this is where you get insertions, deletions, etc. that can cause things like frameshift mutations.
Basic 5 steps of a Southern Blot and how to interpret results
1) Chop up / cleave DNA of interest 2) Run it on gel electrophoresis to separate the DNA pieces based on size and charge. 3) Transfer the gel onto a filter; fragments stay separated. Filter is good because it's stronger and better defined. 4) Incubate blot with many copies of a probe, which is single-stranded DNA complementary to the gene/sequence of interest since you're seeing if that gene is in this sample of DNA. This probe will form base pairs with its complementary DNA sequence and bind to form a double-stranded DNA molecule. The probe is either radioactive or has an enzyme bound to it (e.g. alkaline phosphatase or horseradish peroxidase) so that it can be visualized. 5) Expose filter to X-Ray film to see results. *Dark spots on X-ray are a positive result for the presence of the DNA sequence / gene of interest. *
1) Normally, p53 is kept low by the protein ___. 2) However, whenever more is needed, ___ and ___ (general enzymes) can attach ___ or ___ (respectively) to keep the enzyme in #1 low.
1) MDM-2 2) Kinases -- phosphorylate Acetyl Transferases (HATs) -- acetylate
How does absence of CFTR produce sxs in Cystic Fibrosis?
No chloride ion, get thick mucous
Where are the two places on a strand of mRNA to which eukaryotic initiation complex can bind?
1. Usually bind to 5' end of mRNA 2. IRES (internal ribosomal entry site) -- name makes sense -allows ribosome to start further in, closer to start codon.
What end of each of the two DNA strands will primers attach to?
3' That way, polymerase will travel 3' to 5', which is what happens during DNA replication, to which this is pretty similar. Also this way, the Polymerase will start synthesizing the 5' end of the new strand first.
Heme prosthetic group: -found on what 2 proteins? -what metal is at the center and what is its charge? How is this charge maintained? -Pyrrole groups and structure
4 Pyrroles coordinating with Iron, each one has a nitrogen that donates electrons to Fe2+ to *prevent it* from becoming Fe3+ so that it can bind oxygen.
Structure of a nuclear pore complex
8 spokes, 1 central channel. Protein filaments extend from the rings, forming a basketlike structure on the nuclear side. GO OVER ANUSHA'S NUCLEAR IMPORT AND EXPORT DIAGRAMS!
phosphatidylethanolamine
glycerophospholipid w/ an ethanolamine head group
phosphatidylcholine
glycerophospholipid w/ choline head group
Rotation around the ___ bond of DNA can lead to different conformations (syn and anti)
glycosidic bond
Turner Syndrome -- what it is and *clinical features* --- "Please remember these clinical features"
A chromosomal disorder in females in which there is only one X chromosome.
Cloning vector
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted. Plasmids are a type of cloning vector. Use these when trying to clone a piece of DNA
Which statements are correct? a) Eukaryotic primary transcripts may contain introns. b) Prokaryotic primary transcripts may contain introns. c) Prokaryotic primary transcripts are not processed. d) The most extensive processing of primary transcripts occurs in eukaryotic mRNAs and in tRNAs of both bacteria and eukaryotes. e) Ribozymes are catalytic RNAs that process RNA.
A- true; primary transcripts are before processing. B- false; prokaryotic primary transcripts do not contain introns. C- false; rRNA and tRNA transcripts are processed in prokaryotes. D- true E- true; many RNAs self-splice, meaning they must have enzymatic properties.
"*STEP questions over this are common*" What transcription inhibitor does each of the following: - (2) inhibits RNA *elongation* by intercalating into DNA template. - (1) inhibits *initiation* of bacterial RNA synthesis by binding to the b subunit of the polymerase. - (1) blocks eukaryotic Pol II and Pol III (higher concentrations) but NOT Pol I or bacterial RNA polymerase. AND how each inhibits transcription, briefly
Actinomycin D & Acridine are planar -- they are intercalating agents -- they *ihibit both eukaryotic and prokaryotic transcription* which is why they are no longer used as ABx. Rifampicin binds to beta subunit in prokaryotes, *preventing initiation of transcription* alpha-amanitin is found in mushrooms and it is very bad for humans, but not bacteria. It shuts down Pol II easily and Pol III in high concentrations, which prevents mRNA and tRNA from being made. Thus, transcription and translation are blocked. Very bad.
3 basic steps of PCR -- what polymerase enzyme builds new DNA starting at the primers?
All of the components are mixed together in one tube in very tiny volumes. 1. *Denaturation* step -- ~95C. To be able to copy it, the DNA needs to be separated into single strands (denatured). This can be done by heating it to over 90C. 2. *Annealing* step -- ~55-60C. Although the temperature is lowered, an excess amount of primers prevents the denatured DNA from reforming the double helix. The primers attach or 'anneal' to their matching sequence on the original DNA strand. 3. *Extension* step -- ~ 72C. *Taq DNA polymerase (Taq Polymerase)* binds to the annealed primer. Taq polymerase works its way along the DNA, replicating the DNA.
Usual location of enhancers and what they are
An enhancer is a short region of DNA that can be bound by proteins (activators) to increase the likelihood that transcription of a particular gene will occur. These proteins are usually referred to as transcription factors. Enhancers are cis-acting. (So transcriptional factors bind to enhancer to increase transcription). Enhancers generally are located at a considerable distance upstream from the promoter region of the gene, but they also may be located within introns or downstream.
Post-transcriptional regulation of RNA -- stability of the RNA transcript
Another way to have one gene that makes an RNA that can then be modified to produce different translational products. The transcript can be degraded if it doesn't need to be translated so that no energy is wasted in translating. In this example, if there is enough iron, the short poly-A tail (shorter the tail, easier to degrade) and degrades the RNA. If not, the IRE binding protein binds to the IRE to protect that tail from degradation and the RNA goes on to produce a protein.
Order of protonation for functional groups on AAs based on low, physiological, and high pH. And relate this to which direction they will go on electrophoresis based on net charge due to surrounding pH (logical).
At *low pH*, the amino acid is protonated at both the amine and carboxylic acid OH. At this pH it carries a net positive charge and can be treated as an acid with two pKa's. At *high pH*, both the carboxyl and amine groups are deprotonated. At these pH values, the amino acid carries a net negative charge and acts like a base. At *physiological pH*, the amino acid is a *zwitterion*, and carries *no net charge*. This is called the isoelectric point of the amino acid (pI). At physiologic pH, the amino acid will be stationary in an electric field (on electrophoresis). At low pH, the amino acid carries a positive charge and will migrate to the cathode. At high pH, the negatively charged amino acid will migrate to the anode. This is the procedure used to analyze and purify amino acids and proteins, called electrophoresis.
What happens in terms of protons when Hgb binds CO2.
At the peripheral tissues, hemoglobin can bind CO2 directly when O2 is released. This reaction produces protons, which contributes to the Bohr effect. CO2 binds, obviously it lowers the affinity of Hgb for oxygen.
Transcription of the lactose (lac) operon in E. coli is *stimulated* by: a) a mutation in the repressor gene that strengthens the affinity of the repressor for the operator. b) A mutation in the repressor gene that weakens the affinity of the repressor for the operator in the presence of low glucose. c) Binding of the repressor to the operator. d) The presence of glucose in the growth medium.
B -- that's the only one that would turn on the lac operon. The others would inhibit the lac operon.
Briefly, how carbon monoxide reduces delivery of oxygen to tissues -- effect on hemoglobin, myoglobin, and cytochrome oxidase.
CO binds hemoglobin 230 times more tightly than oxygen, and causes oxygen at other sites to bind with greater affinity, reducing O2 delivery Carbon monoxide also binds to myoglobin with 60 times greater affinity than that of oxygen, and impairs its ability to utilize oxygen. This causes reduced cardiac output, which may result in brain damage. Carbon monoxide binds to cytochrome oxidase interfering with aerobic metabolism and efficient ATP synthesis. Cells respond by switching to anaerobic metabolism causing lactic acidosis and eventual cell death.
Post-transcriptional regulation of RNA -- multiple poly-A tails and getting different end products.
Can see in image example that there are two poly-A tails. The cleavage spot is different in the thyroid and the brain, giving you different mRNAs and thus different final proteins. NOTE: This is not a polycistronic gene -- those have multiple genes each with their own shine-delgarno sequence, start codon, and stop codon. This is all on the same gene.
Cis-acting vs Trans-acting factors/DNA sequences/elements
Cis-acting -- Cis = same -- these must be on the same chromosome as the gene in question. Include promoters, enhancers. Trans-acting -- can be on other chromosomes and still affect a gene.
The majority of p53 mutations in cancer pts occur in what part of the p53 protein?
DNA binding domain
helix-turn-helix DNA binding motif and some other DNA binding motif names that you only need to recognize.
DNA binding motif = the part of proteins that actually binds to the DNA. A protein domain composed of two α helices joined by a short strand of amino acids and is found in many DNA binding proteins, INCLUDING THE LAC REPRESSOR! Binds to the *Major Groove*. If you see this or any of the other ones, you should think "ah okay, that protein can bind to DNA!"
What enzymes are involved in DNA replication?
DNA helicase and DNA polymerase
DNA vs RNA stability
DNA is more stable because of the phosphodiester bond RNA is less stable than DNA. Sensitive to basic conditions -- OH good nucleophile that attacks H of OH.
DNA Slippage (of DNA Polymerase) and Frameshift mutations.
DNA replication within a run of a single base can produce a single base frameshift. In this example, a run of five As is replicated and, depending on whether a slippage occurs in progeny strand or template strand, a T may be added or deleted from the DNA. Polymerase fails to synthesize complementary T.
Western blot
Detects *proteins*. Used for HIV. The intensity of the signal should correlate with the abundance of the antigen on the membrane. Uses *antibodies* as markers
variable expression (genetics) def and possible causes
Different degrees of clinical expression (like differences in severity) with the same genotype Possible causes: Environmental factors, modifier genes.
allelic heterogeneity
Different mutations in the same locus produce the same phenotype hetero because referring to "gen" - beginning -- locus
At which level(s) does eukaryotic gene expression occur? a) Translation b) postranscriptional processing c) Transcription d) posttranslational processing e) all of the above f) none of the above
E - all of the above
Pros and Cons of Microarray / Comparative Genomic Hybridization (CGH) test
First Disadvantage -- means it can tell you if and where something is missing, but cannot tell you further details. *It cannot read the spelling of DNA -- so do not use this to determine if there are mutations* -- only chromosomal abnormalities.
4 main differences between prokaryotic and eukaryotic gene regulation
First bullet point -- eukaryotic genes are off and need to be turned on; prokaryotic is the opposite.
Flippase and Floppase and membrane asymmetry
Flippase: moves phospholipids in Floppase: Moves non-specific lipids out Both use ATP Maintains asymmetry of lipids on each side of membrane.
3 things needed to form ribosomes Once these are transported to the cytoplasm, what is formed?
Formation of ribosomes requires assembly of the *45S pre-rRNA* with *ribosomal proteins* and *5S rRNA* (which is synthesized elsewhere in the nucleolus). Pre-ribosomal particles are then exported to the cytoplasm, yielding the 40S and 60S ribosomal subunits.
Nonpolar amino acids are
GLAM VIP
Germline vs Somatic mutations
Germline: -located in one of reproductive cells (sperm or egg) -All cells are affected. So no matter where you take a cell sample from, have that mutation. Somatic: -Mutation developed sometime later in embryological development. Sample only the affected region; not in all cells. -Cancer is the best example of this. e.g., liver cancer, need liver biopsy -- can't just take a hair.
E
Glutamic Acid
Condensing of DNA into chromatin -- how much does it shorten the DNA?
If not shortened, each cell has ~2m of DNA Packaging of DNA with histones yields a chromatin fiber approximately 10 nm in diameter, which shortens its length about sixfold. It is further condensed by coiling into 30-nm fibers, resulting in a total condensation of about fiftyfold.
How should ABx that inhibit transcription and translation be used?
If one doesn't work, try an antibiotic that targets the other process. that's what test questions will want you to recognize
Anti vs Syn conformation in pyrimidines, purines, and guanosine 5'-phosphates
In pyrimidines, steric clashes between the sugar and the O2 of the base strongly disfavors the syn conformation. In purines, the anti and syn conformations readily interconvert, with anti being more stable in most cases. The syn conformation is stabilized in guanosine 5′-phosphates because of favorable interactions between the 2-NH2 group and the phosphate oxygens.
Formation of Prokaryotic Initiation Complex What type of energy is used to do this?
Initiation factors bind to the small ribosomal subunit This helps the RNA bind IFs scan for the Shine Delgarno sequence This positions the AUG to fall into the P site of the ribosome. fMET tRNA comes in Large ribosomal subunit comes in and forms a cap. *GTP* is used (don't worry about the text in that slide, or numbers of IFs, etc. Just know what I typed)
Inosine in anticodons and what nucleotides can it bind to
Inosine is commonly found in tRNA anticodons and is essential for proper translation of the genetic code in wobble base pairs. it most closely resembles guanine and it can base pair/H-bond with either A, U, or C (*mnem: UCA*) which gives the tRNAs more flexibility. Part of wobble hypothesis because it pairs with 3rd nucleotide of codon and can produce the same amino acid regardless of whether it binds to A, U, or C in many cases.
Our genetic code is degenerate. What does this mean? What is the implication of this (hint- silent mutations).
It means that multiple codons code for the same amino acid in most cases. Like in picture, there are 4 codons that make proline. But only AUG (start codon) makes methionine. Degenerate code allows certain mutations to still code for the same amino acid. "silent" mutations―different nucleotide in DNA but same amino acid in protein
Formation of eukaryotic initiation complex Include the Kozak sequence
Kozak sequence is the equivalent of the shine-delgarno sequence in proks. However, whereas the SD sequence was before the AUG start codon, the Kozak sequence *surrounds* the start codon in euks. small ribosomal subunit will bind here
What is the purpose of Fluorescent in-situ hybridization (FISH) / what can it tell you?
Labeled chromosome-specific DNA segment (probe) is hybridized with metaphase, prophase, or interphase chromosomes and visualized under microscope Commonly used to determine *if portion of chromosome is deleted/duplicated.* e.g., LSI Williams disease is caused by a missing piece of a chromosome, so if it is suspected, can order a FISH to see if that's the case. This is one of the three "cytogenetic" tests, which focus on chromosomes themselves.
If it asks you the likely recognition sequence for a restriction enzyme
Look for a palindrome -- e.g., AAGCTT (that was the quiz answer). Palindrome --- 5'-->3' same for both strands.
More __ = higher melting point
More GC = higher melting point
Liquid Chromatography -Modern day most common way to determine what? -separates proteins based on ___ -Which peptide fragments will elute first? -why are proteins degraded to peptides first? -how do you identify/characterize them afterwards?
Most common way to determine a person's proteome. separates proteins (technically, they're peptides) based on *polarity* and then analyzes them with mass spec. Protein fragments with more polar AAs will be eluted first as they are in the mobile phase because it makes them easier to ionize for mass spec. This is how you separate them. Then you characterize/identify them with *mass spec*.
Structure of 5' cap (need to be able to recognize it)
ONLY the part in blue 7-methylguanosine so it's just a guanosine with a methyl on Nitrogen at position 7.
How to read results of dideoxy sequencing
Once these reactions are completed, the DNA is once again denatured in preparation for electrophoresis. The contents of each of the four tubes are run in separate lanes on a polyacrylmide gel in order to separate the different sized bands from one another. After the contents have been run across the gel, the gel is then exposed to either UV light or X-Ray film, depending on the method used for labeling the DNA. IMAGE: This is a polyacrylmide gel of the reactions in the "G" tube (the same sequences seen in figure above). The longer fragments of DNA traveled shorter distances than the smaller fragments because of their heavier molecular weight.The blue section indicates the primer, the black section indicates the newly synthesized strand and the red denotes a ddGTP, which terminated the chain.
Post-transcriptional regulation of RNA -- Alternative splicing in Drosophilia sex determination
One splice pattern gives you a non-functional tra protein, making the fly a male. The other gives you a functional tra protein, making the fly a female. In male pattern, blue has a stop codon in it that prevents tra from being made if it isn't spliced out.
Overview of similarities and differences between DNA replication and transcription of DNA
Other similarities: -in both, DNA must be de-condensed first (removal of supercoil) Other differences: -Transcription does not require a primer; DNA Pol attaches to the primer in replication. -*In eukaryotes, TATA box is at -30, whereas its at -10 in prokaryotes.* -Eukaryotes have 3 types of RNA polymerase (RNA Pol I, II, and III which make the 3 types of RNA) whereas bacteria just have the one RNA Polymerase. -Prokaryotic RNA Pol is already assembled before transcription; in eukaryotes, it has to be assembled starting with TATA Binding Protein (TBP) binding to the TATA box at -30.
Effect of pH on oxygen binding to hemoglobin -- Bohr effect -high pH vs low pH; lungs vs. tissues -when does the curve shift left and right? -role of protons -In tissues, increasing [H+] forces the equilibrium which way?
Oxygen binds well at higher pH Oxygen is released at lower pH Blood in lungs has higher pH (fewer H+) than blood in capillaries of metabolic tissues Curves shift to the right under more acidic conditions - toward lower oxygen affinity At a single pressure, if you change pH, you can see that oxygen affinity changes. Protons are released when Hgb binds oxygen (T --> R) To the left, according to the law of mass action, toward increasing concentrations of deoxy-Hb and release of O2
p and q arms of a chromosome
P stands for petite; Q was jut the next letter in the alphabet. Likely to see chromosomes labeled with this; e.g., Xp = p arm of X chromosome.
Phospholipase C cleaves ___ to ___ and ___, which are second messengers involved in the action of some hormones
PIP2 --PLC--> IP3 + DAG Phosphatidylinositol 4,5-biphosphate inositol triphosphate (IP3) diacylglycerol (DAG)
The binding sites on DNA for regulatory proteins (repressors and activators) have what feature in their sequence/spelling? including the lac operon site. Also cooperativity in protein binding
Palindromes! Like seriously, likely to be a test question. Regulatory proteins will have palindromic sequences, ex: GAATTC Cooperativity -- one part of the protein binding occurs readily, 4 parts binding is even faster and stronger.
Phosphate groups can be enzymatically added to and removed from the hydroxyl (-OH) side chains of what 3 Amino Acids? This is a common and very important means of regulating protein function.
Phosphate groups can be enzymatically added to and removed from the hydroxyl (-OH) side chains of *serine, threonine, and tyrosine*
What is an operon? Also polycistronic RNA and proks vs euks
Polycistronic RNA means one RNA Pol can encode several genes. This is only present in proks. The series of genes is called the operon and each gene has its own shine-delgarno sequence, start codon, and stop codon. This allows easier control over a group of genes that are part of a related function/end goal --- the best example is the lac operon.
In a person with cancer, you would expect what levels of p53?
Probably absent, or a mutation in the p53 gene. It's elevated in healthy people after DNA damage though because it tries to repair it. Without it, tumors can grow freely.
Process affected (in translation) and site of action of: *Tetracyclines* -- including what site on the ribosome
Process affected: *Binding of aminoacyl-tRNA* Site of action: both proks and euks, small subunit --- *30S (proks) or 40S (euks) subunits*
Process affected (in translation) and site of action of: *Cycloheximide*
Process affected: *Elongation* Site of action: *80s subunit of euks*
Process affected (in translation) and site of action of: *Puromycin*
Process affected: *Peptide transfer* Site of action: *70s or 80s subunits* (large subunit of either proks or euks)
Process affected (in translation) and site of action of: *Erythromycin*
Process affected: *Translocation* Site of action: *50s subunit* of proks
Process affected (in translation) and site of action of: *Fusidic Acid*
Process affected: *Translocation* Site of action: *Elongation Factor G of proks*
Process affected (in translation) and site of action of: *Streptomycin*
Process affected: *initiation and elongation* Site of action: *30S of proks* (makes sense; antibiotic so only for proks)
Process affected (in translation) and site of action of: *Neomycins*
Process affected: *translation* Site of action: many; proks
Process affected (in translation) and site of action of: *Ricin*
Process affected: multiple Site of action: *60s subunit of euks*
Shine-Dalgarno sequence
Prokayotic initiation = Shine-Dalgarno sequence In prokaryotes, what is the name of the RNA sequence that ribosomes bind to, thus initiating translation. Ribosomes bind at this sequence, which is upstream from the start codon (AUG/methionine) so that the start codon is the first codon translated.
Some proteins can fold on their own while others need some help. How do *chaperones* and *accessory factors* help with the folding of some proteins?
Proteins that facilitate folding include: *Accessory Factors* Modify specific amino acid side chains to alter the outcome of protein folding. *i.e.,* it goes to particular AAs and alters their R group in some way, which is how they manipulate how the protein will fold. *Chaperones* Do not change the final outcome of the folding process, but prevent protein aggregation prior to completion of folding and prevent formation of non-productive intermediates. *i.e.,* chaperones bind to specific parts of proteins in a way that prevents them from folding into undesired shapes. Called *heat shock proteins* because as temp increases, they start being synthesized more and more to try to keep the protein folded.
Proteome
Proteome = all proteins (analogous to genome) Number of unique proteins probably exceeds 250,000 Proteomics is the science of determining which proteins are produced in a cell or tissue under a specific set of conditions (disease, differentiation, development, stress, aging, drug treatment) The proteome is a functional representation of the genome. In general, proteins in a complex mixture are first separated, then characterized - ideally, no proteins are lost during separation
__ and __ are aromatic and planar structures
Purine and Pyrimidine
3 steps of poly-A tail addition in RNA processing. Include *endonuclease* and *polyadenylate polymerase* (Pol A Pol) Transcription is terminated when RNA Pol reaches the ___ ___ ___.
RNA Pol reaches *cleavage signal sequence*, terminating transcription. Endonuclease knocks off 3' end, leaving you with free 3'OH Polyadenylate polymerase makes the poly-A tail by adding a bunch of Adenosines to 3' end.
Type 2 Restriction Endonucleases -*what is the recognition sequence going to be?* -*where on the DNA do they bind?* -what they do / their function/goal -found only in what kind of organisms? -why are these enzymes preferred over other enzymes? -do these Type 2 restriction endonucleases require ATP?
Recognition Sequence- has to be a *palindrome* (e.g., AAGCTT) ; (it recognizes palindromes on the DNA) Bind *at the recognition site*, not before or after it. They scan the DNA for specific recognition sites and cleave there. They are enzymes within *prokaryotes* that protect the bacterium from foreign DNA (viruses). And of course this makes sense because DNA experiments are on bacteria, not eukaryotes, most of the time. Overall function: *cleave foreign DNA into smaller fragments*, which is a necessary step before cloning. Restriction endonucleases are preferred because they cut DNA at specific places, so you get control over the experiment. Other DNA cleavage enzymes cut up DNA more randomly. Type 2 Restriction Endonucleases are the best option for cleaving the DNA molecule at a few precisely-located sites *so that a small set of homogeneous fragments are produced.* Use recognition site, do not need ATP, but usually do need a cation like Mg2+.
4 basic steps of DNA excision repair
Recognize damaged DNA Remove it by excising part of one strand Resynthesize DNA to fill gap using genetic info from other strand Ligate to restore continuity of DNA backbone Base and Nucleotide excision repair
RPA (replication protein A)
Single Stranded Binding protein in eukaryotes (keeps strands separated after helicase separates them)
fusion of two acrocentrics results in what type of chromosomal translocation? How this works: two pieces (one section of each chromosome) from short side of centromere join, and two long sections of chromosomes join. This leaves you with one large pair of chromosomes and one tiny pair of chromosomes) (acrocentric = when chromosome's centromere is located quite close to the end of the chromosome)
Robertsonian translocation (other two types are balanced and unbalanced, which you know. all translocations are bad. balanced translocations often result in miscarriage/unsuccessful attempts to get pregnant and 10% of children with chromosomal abnormality have a parent with a balanced translocation -- so you need to offer testing to the parents of these kids).
alpha helix: -which way do R groups point? -how many residues/AAs per turn? -stabilized by __bonds between __ and __ that are how many AAs away? -rarely has what AA?
Rod-like, tightly coiled R groups extend outward 3.6 residues per turn Stabilized by H-bonds between carbonyl oxygen and amide group *4* residues away Usually right-handed (turns clockwise from N terminus Prolines are the most rare because they cause a kink in the protein. Glycines are the second rarest because they're small and disrupt protein structure. Interaction between amino acid residues can either stabilize (e.g. GluxxxArg) or destabilize (e.g. adjacent charged or adjacent bulky residues)
polar amino acids
SAG CT serine, threonine, asparagine, glutamine, cysteine
Northern Blot
Similar technique to Southern, except that Northern blotting involves radioactive DNA probe binding to sample RNA. It looks at *RNA*! Northern blots allow investigators to determine the molecular weight of an mRNA and to measure relative amounts of the mRNA present in different samples. Steps: 1) RNA (either total RNA or just mRNA) is separated by gel electrophoresis. Because there are so many different RNA molecules on the gel, it usually appears as a smear rather than discrete bands. 2) The RNA is transferred to blotting paper. The RNA molecules retain the same pattern of separation they had on the gel. 3) The blot is incubated with a probe, which is single-stranded DNA. This probe will form base pairs with its complementary RNA sequence and bind to form a double-stranded RNA-DNA molecule. 4) The location of the radioactive probe is revealed by looking for color on an X-ray
Skewed Lyonization / Skewed X inactivation in females? Manifesting female carrier.
Skewed X chromosome inactivation occurs when the inactivation of one X chromosome is favored over the other, leading to an uneven number of cells with each chromosome inactivated. So this is when it's not 50/50 between different cells on which X chromosome was inactivated. One X chromosome more favorable for some reason so the less favorable X chromosome is inactivated more than the favored one in the female. A mutated gene could be on the favored X chromosome, meaning this may result in signs of the disorder because she would not be producing enough of the protein necessary for the body's normal functioning, and she would be referred to as a *manifesting female carrier*. These *manifesting female carriers* are similar to the male condition where a mutation on an X chromosome is likely to be active because the Y chromosome can't balance it out. The mutated gene is abnormally activated and they can get the condition.
In response to some hormones, Phosphatidylinositol 4,5-biphosphate (PIP2) is phosphorylated to ___ by the enzyme ___.
Some hormone (including insulin) induce a signaling cascade where phosphatidylinositol-4,5 biphosphate (PIP2) at the plasma membrane is phosphorylated by the enzyme PI-3 kinase to phosphatidylinositol-3,4,5 triphosphate (PIP3)
Epigenetics
Something that alters gene expression but doesn't change the DNA sequence (e.g., methylation)
Spur cell anemia - relationship with cholesterol (cause)
Spur-cell anemia is an uncommon type of haemolytic anemia that occurs in patients with advanced liver failure. The mechanism responsible for formation of spur cells has been attributed to changes in the composition of lipids of the red cell membrane, with *increased cholesterol content and increased membrane rigidity. This alters the shape of the cells and makes them more susceptible to trapping and destruction by the spleen. *
** Steps of protein export from the nucleus to the cytoplasm 1) Proteins bound for export contain AA sequences also called __ __ __. 2) The sequence is recognized by ___, which binds it and carries it to the cytoplasm. 3) These proteins that carry the protein bound for export rely on ___, which binds to it in the nucleus. 4) Then what happens to the cargo protein in the cytoplasm?
Start this video at 0:54: https://www.youtube.com/watch?v=Q7F2vGwfsL8 1) Proteins bound for export contain AA sequences called *nuclear export signals (NESs)* 2) *Exportins* (note "export") recognize NESs to direct the proteins from the nucleus, and out to the cytoplasm. 3) Exportins also form complexes with *Ran/GTP* in the nucleus. 4) In the cytoplasm, GTP hydrolysis and release of Ran/GDP leads to dissociation of the cargo protein.
Signal specific trans-acting factors -- steroids -- what is their effect on transcription? Include zinc fingers -- what is their role?
Steroids, when bound to their intracellular receptors, usually function as transcriptional *activators*, but *can also be repressors.* Steroid comes across membrane, attaches to receptor (which is floating inside of the cytoplasm -- not a trans-membrane receptor), crosses nucleus, binds to receptor site on DNA to promote transcription *zinc fingers* make up part of the steroid receptor and allow the complex to interrogate the DNA to find the right place to bind. After this, the promoter is activated and transcription can begin.
phosphatidylserine
Synthesized from phosphatidylethanolamine; has a negative charge
Contribution of His to stability of α and β subunits of T-hemoglobin. (subunits = different proteins/polypeptide "chains" -- term he uses) - The T (deoxygenated) form of hemoglobin has more ___ between different chains. For example... - The C-terminus of one of the His residues (called ___ , aka ___), located on the G α-helix of the β subunit, forms a H-bond with *what AA* on the adjacent α subunit? -The C-terminal of this His is also H-bonded with *what AA* in the ___ corner of the same β subunit.
T-hemoglobin (deoxygenated) is more compact and stable because of these interactions. - salt links (/ H-bonds) - *His HC3* (aka His 146), located on the *G* helix of the β subunit, forms a *hydrogen bond* with *Lysine* on the adjacent α subunit. [blue circle in image] -It also forms a *hydrogen bond* with *Aspartic Acid* (Asp) in the *FG corner* (corner between F and G α-helices). [maroon circle in image]
What forms at the end of a chromosome?
T-loop
Telomerase
Telomerase puts telomeres on the ends of DNA strands to finish them once primers don't have room to attach. (lagging strand)
Genetic conditions in which there are more than two X chromosomes: -Terminology -What tends to be impaired the most with the addition of extra X chromosomes?
Tend to see 15 point IQ drop with each extra chromosome; more X chromosomes leads to greater intellectual problems. # of x chromosomes then "-somy"
When is His 146 (HC3) protonated and deprotonated? When protonated, it forms a ___ bond with ___
The R groups of Hgb change proton numbers based on whether oxygen is bound. His HC3 is a major player in these proton changes. *In the T form, His146(β) is protonated because it forms an ion-pair (salt bridge) with Asp94.* About 50% of released protons come from the two His146 residues, the rest from other acidic groups that similarly change their pKa values as the conformation changes.
What is a splice donor and acceptor? What's the branch site? (*mnem*) Where are the splice donor and acceptor sites located on an intron? (hint -- donor vs acceptor defined by what end of RNA it's on)
The branch site is a *conserved sequence* (always stays same) that allows snRNPs to recognize splicing locations. D5 mnem: Triple AAA car insurance -- 3 A -- 3' is acceptor
How is the lac operon an inducible system?
The lac operon is an example of an inducible system. This operon is always turned off unless an inducer -- lactose -- is available from the environment; lactose triggers the expression of genes in this operon.
Other than the anticodon, variation amongst different tRNA molecules that helps them get recognized
green and orange differ between specific AA tRNAs
How is the ribosome a ribozyme (how is it an enzyme)?
The part of the ribosome that translates DNA to protein (its catalytic center) has no proteins (enzymes) in it. Therefore, the ribosome itself must be an enzyme since ribosomes definitely do translation.
DNA strands are: (5') CGCTATAGCGTTT (3') (3') GCGATATCGCAAA (5') (or it could only have one strand listed) What will be the mRNA sequence?
The real key to this is knowing that RNA Pol travels to the right along the 3' --> 5' strand and spits out RNA in the 5' to 3' direction, so the RNA (the answer) will be 5' to 3' and have conjugate base pairs t the 3' to 5' strand of DNA (with U instead of T). Forget the 5' to 3' DNA strand. The question may or may not have both strands written out, but you can determine answer from either. Also, if 5' and 3' ends of DNA not indicated, assume it's written 5' --> 3'. *The mRNA sequence will be identical to the 5' --> 3' strand, but replace T's with U's.* Answer: (5') *CGCUAUAGCGUUU* (3')
Cisplatin
Used in chemotherapy Wrecks DNA...causes so much damage that replication cannot occur (so you would target cancerous cells) Forms cross links that can't be easily broken down.
What happens to a stem cell when it is placed in a tissue that really needs more cells (as its cells cannot replicate on their own)?
The stem cell itself does not carry out the functions of the cells in that tissue. It produces cells that can become any kind of cell, and they choose what type of cell to be and start replicating.
Summary of how H+, CO2, and BPG affect the affinity of Hgb for Oxygen.
Three allosteric effectors, H+, CO2, and BPG, cause a decreased affinity (increase in P50 value) of Hb for oxygen.
Post-transcriptional regulation of RNA -- regulation of translation itself if it has already started.
This is a way to prevent or limit translation after ribosome has already recognized the RNA. In image, you can see that little proteins (translational repressors in picture) can bind and prevent poly-A tail / 3' end from binding to proper site on ribosome, preventing translation. Another thing that can happen is these proteins can bind to 3' untranslated region, and ribosome can translate up to that point but can't finish bc it's blocked.
How do the three α-chains of a collagen molecule interact (kinda looks like a piece of candy)
Three α-chains of a collagen molecule wind around each other to form a superhelix Collagen helix (polyproline type II helix or α-chain) is unique, quite distinct from the α-helix, and is *left-handed* with *three amino acids per turn*. Three separate α-chains are twisted about each other, in a *right-handed sense*, to form a superhelix with the *glycines in the shared interior*.
How to determine the ratio of His/HisH+ in AAs.
To determine the ratio of His/HisH+ (conjugate base/acid) we subtract the pKa from the pH; then raise both sides to the 10th power to get rid of the log to yield 10^(pH-pKa) = [His/HisH+] or [A-]/[HA]
What releases the supercoiling of DNA during *transcription* and *replication*
Topoisomerase
What enzyme relieves torsional strain during DNA replication?
Topoisomerase cuts it and reseals it
At what stage of DNA ---> Protein is it most efficient to regulate a gene (like to prevent it from being expressed)
Transcription because if you can stop it there, you don't waste any energy in converting DNA to RNA or later steps.
Transcription occurs at clustered sites called ___.
Transcription occurs at clustered sites called *transcription factories*.
What is the main problem with iron being used to transport heme?
Transition metals, such as iron and copper, readily bind oxygen, but free iron can produce reactive oxygen species, such as hydroxyl radicals, that damage biological macromolecules. (but it's needed to transport heme b/c heme is not soluble in blood and does not readily pass into tissues on its own)
Transition point mutation vs Transversion point mutation
Transition point mutation, purine for purine (A for G or G for A) or pyrimidine for pyrimidine (T for C or C for T) Transversion point mutation, purine for pyrimidine or vice versa
Patau syndrome and clinical features and prevalence
Trisomy 13 median life expectancy is 7-10 days Clinical features: 1. Cleft lip / palate 2. Severe mental retardation 3. Microphthalmia 4. Polydactyly 5. Microcephaly 6. Congenital heart disease 7. Etiology - nondisjunction or inherited translocation Incidence = 1/5000
Edward Syndrome
Trisomy 18 median life expectancy is 4-14 days 1. Severe growth deficiency 2. Mental retardation 3. Clenched fist with overlapping finger 4. Rocker bottom feet 5. Congenital heart disease 6. Etiology - nondisjunction or inherited translocation Incidence = 1/3000
W
Tryptophan (Trp)
What is p53? (what kind of molecule) What 2 main functions (4 total) does p53 have?
Tumor Suppressor Gene p53 is a DNA-binding protein that is present in low levels in normal cells but strongly elevated after DNA damage where it either promotes cell cycle arrest or apoptosis, largely through acting as a transcriptional regulator Really, just know that p53, after DNA damage, will promote either *cell cycle arrest* or *apoptosis*
Y
Tyrosine
Blue-white selection -used to determine which cells underwent ___. -interpreting results: white colonies vs. blue colonies.
Used to determine which cells underwent *recombination*; answers "Which cells contain *recombinant vs. nonrecombinant DNA*?". *White Colonies* - contain what you're looking for -- Cells transformed with vectors containing *recombinant* DNA will produce white colonies *Blue Colonies* - cells transformed with non-recombinant plasmids (i.e., only the vector) grow into blue colonies. (Insert DNA into bacterial pUC plasmids; the pUC plasmid has the gene that encodes B-galactosidase [so the pUC plasmid contains gene that encodes the marker -- blue vs white]).
How does a lack of vitamin C lead to scurvy
Vitamin C is necessary for the hydroxylation of proline and lysine residues into hydroxyproline and hydroxylysine during collagen synthesis. No vitamin C, no collagen made, or at least a lack of it. Collagen containing insufficient hydroxyproline and hydroxylysine loses temperature stability and is less stable than normal collagen at body temperature. The resulting clinical manifestations are distinctive and understandable: suppression of the orderly growth process of bone in children, poor wound healing, and increased capillary fragility with resultant hemorrhage, particularly in the skin.
The lac operon of E. coli, in a nutshell
Want to turn off lac operon if there's glucose available; if no glucose available, turns on lac operon so that lactose can be broken down and used for energy. 1) The lac operon of E. coli contains genes involved in lactose metabolism. It's expressed only when lactose is present and glucose is absent. 2) Two regulators turn the operon "on" and "off" in response to lactose and glucose levels: the lac repressor and catabolite activator protein (CRP/CAP). 3) The lac repressor acts as a lactose sensor. It normally blocks transcription of the operon, but stops acting as a repressor when lactose is present. The lac repressor senses lactose indirectly, through its isomer allolactose. 4) CAP/CRP acts as a glucose sensor. It activates transcription of the operon, but only when glucose levels are low. CAP senses glucose indirectly, through the "hunger signal" molecule cAMP.
consequence of inverted repeats
When DNA is inverted, cruciforms can form and disrupt DNA structure (inverted repeats = palindromes)
** Changes in Hgb's shape when O2 binds (T -> R state) #3 Movement of Fe and cooperativity
When one oxygen binds, the Fe moves more to the center of the porphyrin rings (image shows it off-center) which allows other O2s to bind better. Caused by the structural change in proximal His.
Lac operon summary
Without lactose in the cell, the repressor protein binds to the operator and prevents the read through of RNA polymerase into the three structural genes. With lactose in the cell, lactose binds to the repressor. This causes a structural change in the repressor and it loses its affinity for the operator. Thus RNA polymerase can then bind to the promoter and transcribe the structural genes. In this system lactose acts as an effector molecule. Lactose is not the preferred carbohydrate source for E. coli. If lactose and glucose are present, the cell will use all of the glucose before the lac operon is turned on. This type of control is termed catabolite repression. To prevent lactose metabolism, a second level of control of gene expression exists. The promoter of the lac operon has two binding sites. One site is the location where RNA polymerase binds. The second location is the binding site for a complex between the catabolite activator protein (CAP) and cyclic AMP (cAMP). The binding of the CAP-cAMP complex to the promoter site is required for transcription of the lac operon. The presence of this complex is closely associated with the presence of glucose in the cell. As the concentration of glucose increases the amount of cAMP decreases. As the cAMP decreases, the amount of complex decreases. This decrease in the complex inactivates the promoter, and the lac operon is turned off. Because the CAP-cAMP complex is needed for transcription, the complex exerts a positive control over the expression of the lac operon.
Chromosomal structural re-arrangements --- 3 diseases
Wolf-Hirschhorn syndrome (4p-) (*mnem* - *W4*) Cri-du-chat (5p-) Micro-deletion syndromes
dideoxy sequencing -- how it works
a method of DNA sequencing (*determining the spelling of DNA*) that uses dideoxyribonucleotides to terminate the growth of DNA strands -- once a ddNTP (dideoxy nucleotide triphosphate) is reached/incorporated, synthesis of DNA ends. Attach primer to DNA, then divide solution into four tubes labeled "G", "A", "T" and "C". Then reagents are added to these samples as follows: "G" tube: all four dNTP's, ddGTP and DNA polymerase "A" tube: all four dNTP's, ddATP and DNA polymerase "T" tube: all four dNTP's, ddTTP and DNA polymerase "C" tube: all four dNTP's, ddCTP and DNA polymerase As the DNA is synthesized, nucleotides are added on to the growing chain by the DNA polymerase. However, on occasion a dideoxynucleotide is incorporated into the chain in place of a normal nucleotide, which results in a chain-terminating event. For example if we looked at only the "G" tube, we might find a mixture of the products in the IMAGE. Once these reactions are completed, the DNA is once again denatured in preparation for electrophoresis. The contents of each of the four tubes are run in separate lanes on a polyacrylmide gel in order to separate the different sized bands from one another.
Plasmids and the three features a plasmid should have if you're cloning DNA
a plasmid is a small, double-stranded, closed-circular DNA strand in a bacteria cell that can replicate independently of the chromosomes, typically in the cytoplasm of a bacterium. Should have these features: 1) *It should be small.* -- The aim of cloning experiments is to isolate the passenger DNA. A small plasmid only contributes a minimal amount of extraneous DNA, thereby making it easier to prepare large amounts of the passenger DNA. Also, it's easier to get smaller pieces of DNA into bacteria than larger ones. 2) *Its DNA sequence should be known.* -- this way, you can manipulate it with the proper complementary DNA strand. 3) *Plasmid vectors should contain a selectable marker that allows cells containing the plasmid to be isolated.* -- e.g., resistance to some antibiotic; something that allows you to separate cells with the plasmid from cells without it.
Which of the following is a DNA sequence? a) repressor b) enhancer c) inducer d) transactivator
a) no -- repressors are proteins b) yes -- this is a sequence within the DNA c) no -- protein d) no -- protein
Which statement(s) is/are correct? a) Chromatin structure can affect the ability to transcribe a particular gene. b) Eukaryotic promoters can be positively or negatively regulated. c) Eukaryotic genes may be regulated by external signals such as hormones.
all are correct
What is a Barr body?
an inactivated X chromosome --- since females have 2 X-chromosomes, one must be inactivated so you don't get double proteins
hemidesmosome
anchors intermediate filaments in a cell to the basal lamina
Acidic (- charged) amino acids
aspartate, glutamate (aspartic acid, glutamic acid)
It is possible to convert the Cys that is a part of Cys-tRNACys to Ala by catalytic reduction. If the resulting Ala-tRNACys were added to a mixture of ribosomes, all the other tRNAs and amino acids, all of the cofactors and enzymes needed to make protein in vitro, and mRNA for hemoglobin, where in the newly synthesized hemoglobin would the Ala from Ala-tRNACys be incorporated? a) Wherever Ala normally occurs. b) Wherever Cys normally occurs. c) Wherever either Ala or Cys normally occurs. d) Wherever the dipeptide Ala-Cys normally occurs.
b) Wherever Cys normally occurs. because Ala-tRNACys is still specific to the cysteine codon; will not recognize alanine's codon.
tRNA anticodon -antiparallel base pairing -- which way is each RNA (t and m) oriented? -tRNA and mRNA pair through what kind of bond?
base pairs to codon in an antiparallel orientation
cDNA library construction -- cloning genes based on their mRNA transcripts.
cDNA = complementary DNA -- a piece of DNA synthesized from an RNA strand using reverse transcriptase (RNA-dependent DNA Polymerase) Why? Because the mRNAs produced had genes in an exon. So you use reverse transcriptase to get the DNA template (I assume) strand. used for genes that are only transcribed in particular cell types (e.g., globulin only made in RBCs) The specific DNA sequences expressed as mRNAs in a particular cell type can be cloned by synthesizing DNA copies of the mRNAs isolated from that type of cell, and then cloning the DNA copies in plasmid vectors.
DNA vs. RNA
deoxyribose sugar vs. ribose sugar (*2'* carbon), thymine vs. uracil , double strand vs. single strand
Porphyria diseases -what they are -all involve accumulation of ___, which is what? -2 categories/types of porphyria -sxs AND main symptom of hepatic porphyria
diseases caused by a disturbance in heme biosynthesis all involve accumulation of *porphyrin*, which is heme without iron. For some reason, there is no iron. 2 categories: hepatic porphyrias and erythroid porphyrias sxs: Excretion of these compounds colors the urine red and accumulation in the skin can lead to extreme photosensitivity. Also associated with increased hair-growth Hepatic porphyria is characterized by severe abdominal pain
Tertiary structure is stabilized by relatively weak interactions which include...(4)
hydrophobic interactions hydrogen bonds salt bridges disulfide bonds
Eukaryotic transcription: TATA box, Invariant (Inr) region
invariant region surrounds the +1 location where transcription begins. In eukaryotes, TATA box is at -30, whereas its at -10 in prokaryotes.
Chromosomal structural abnormality: A chromosome with two identical arms is called a ___
isochromosome
3 examples of single gene diseases (*mnem*)
mnem: *SMH* at these single gene diseases Sickle cell anemia Marfan syndrome Huntington's Disease
Polycistronic genes are only found in ___.
prokaryotes (remember, example is lac operon; these are genes transcribed together that each have their own shine-delgarno sequence, start codon, and stop codon; but I guess they are regulated together like the lac operon) Polycistronic genes have *one promoter* for all of the genes in that operon. Assume each individual gene has its own everything else.
Elongation (Translation) in both proks and euks -EF Tu and GTP -where does the second tRNA bind? -what is used for energy? -how does the protein/polypeptide start to form? Enzyme? -what happens after that?
same for both proks and euks (says fMet in diagram, but that's only for proks) also, the second one that comes in at the bottom should not have an fMet! fMet is only used during initiation and then never again. The protein starts to form when *Peptidyl Transferase* makes a peptide bond between the first two AAs. It keeps making this bond btw the AAs in the P spot and in the peptide chain. After that, (1) The ribosome moves one codon toward the 3' end of the mRNA (*translocation*). (2) The growing peptide is now in the P site, leaving the A site open for the next aminoacyl-tRNA. (3) The uncharged tRNA dissociates from the E site.
Passive vs selective transport across the nuclear envelope
selective requires energy -- proteins and RNA mainly
process in which a part of one chromosome is moved to another chromosome, leading to a chromosomal abnormality (e.g., part of chromosome 9 moved to chrom 22 and part of chrom 22 moved to chrom 9)
simple re-arrangement (I guess this is also called balanced translocation)
RNA structure
single stranded 3' OH --- P --- 5' OH Phosphate joins the 3′-OH group of one ribose with the 5′-OH group of the next ribose. This linkage produces a polyribonucleotide having a sugar-phosphate "backbone." Purine and pyrimidine bases extend away from the axis of the backbone and may pair with complementary bases to form double-helical base-paired regions.
Splicing of introns requires ___, which are made of ___ and ___. It's in the name!
snRNPs (small nuclear ribonucleoprotein particles: RNA + protein). It's in the name!
Splicing process. snRNPs, splice donor, splice acceptor, and how intron is removed. What is a lariat?
snRNPs splice at 5' (donor) site, once they recognize branch site. Then they bring 5' end over to 3' end (donor to acceptor) so everything in between (the intron) is removed and the two exons are stitched together. A *lariat* is formed during eukaryotic splicing. Just know that it's the loop and what process it's a part of.
Mutation vs. Polymorphism
so it has to meet two requirements: change in DNA + occurs in at least 1%.
what happens if there's one insertion and one deletion mutation?
some codons are still changed, but actually not as bad as if there's just one of them because at some point, the reading frame is restored and you get your original reading frames. But even a couple of codon changes is still very bad.
Role of hydroxyproline and hydroxylysine in collagen. These are dependent on __ and __. What enzyme makes hydroxyproline and what series of 3 AAs does it recognize?
the conformational change to hydroxy for proline and lysine residues really makes collagen take its shape. dependent on vitamin c and ferrous iron prolyl hydroxylase recognizes Gly-X-Proline
-Basic steps of prokaryotic transcription (DNA to RNA) -- how is it started and how does the RNA Polymerase holoenzyme become just the core enzyme?
the sigma subunit acts as a brake once it gets to promoter, so it needs to be removed for RNA Pol to be able to continue on along the DNA strand and start transcribing Holoenzyme vs Core Enzyme -- whether or not sigma subunit is bound.
AA and IVC spacial relationship in terms of above/under
this image is a coronal US view IVC over AA (think of it as normal superficial positions of veins)
autosomal recessive
two copies of an abnormal gene must be present in order for the disease or trait to develop this isn't de novo because it's not dominant afterwards. De novo is autosomal dominant because it acts dominant afterwards.
Polygenetic inheritance
two or more genes contribute to the phenotypic expression of a single characteristic
α-thalassemia (disease) is an example of
what can happen from mutations as the codons are changed
- Chromatin is composed of ~200 base pairs of DNA, 147 of which wrap around the histone core. The histone core with DNA wrapped around it forms a ___ ___ ___. -H1 histone does what -all of this forms a ___
~*nucleosome core particle* -Histone H1 is bound to DNA where it basically holds everything together. -This forms a *chromatosome.* (This is how chromatin is formed)