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What is the mechanism of translation?

1. Chain Initiation: To begin, you need to form initiation complex, basically an assembly of everything. This includes mRNA, initiator tRNA (fmet), and ribosome (initiation factors, and GTP aids in formation of initiation complex). The initiation complex forms around initiation codon (AUG), which is just downstream of the Shine-Dalgarno sequence. The Shine-Dalgarno sequence is the "promoter" equivalent of translation for prokaryotes (Kozak sequence for eukaryotes). o 2. Chain Elongation: protein made from N terminus to C terminus. mRNA codons read from 5' to 3' end. Elongation consists of: § 1. Binding: new tRNA with its amino acid (tRNA + amino acid is called aminoacyl- tRNA) enters the A site. GTP and elongation factor required. § 2. Peptidyl transfer: attachment of the new amino acid to existing chain in P site. The already existing chain in P site migrates and attaches to the aminoacyl-tRNA in the A site. § 3. Translocation: the lone tRNA in the P site gets kicked off (E site), and the tRNA in the A site, along with peptide attached to it, moves into the P site. The mRNA gets dragged along also - the codon that was in the A site is now in P site after translocation. A site is now empty and ready for the binding of a new aminoacyl-tRNA to a new codon. Elongation factor and GTP required. o 3. Chain Termination: When a stop codon is encountered, proteins called release factors, bound to GTP, come in and block the A site. Peptide chain gets cleaved from tRNA in the P site. Peptide chain falls off and the whole translation complex falls apart. o Amino acid activation: enzymes called aminoacyl-tRNA synthetases attach the correct amino acids to their corresponding tRNAs. ATP required. • Post-translational modifications to proteino Modifications of the protein through addition of groups to the protein through covalent bonds or cleavage of the proteino Deals with activation/inactivation or enhancing the protein's functiono Examples include phosphorylation, glycosylation, and ubiquitination (inactivation by tagging protein for proteasome degradation)

Cystine

2 cysteines that have formed a disulfide bond

Tertiary structure

3-D structure stabilized by hydrophobic interactions, acid-base interactions (salt bridges), hydrogen bonding, and disulfide bonds Depends on distant group interaction • Stabilized by hydrogen bonds, van der Waals, hydrophobic packing, disulfide bridge formation. Disulfide bond formation happens on the exterior of the cell (covalent bond of two cysteines) Extracellular space prefers the formation of disulfide bonds (the oxidizing environment.) Hydrophobic interactions and polar interactions between side chains

What does denaturation do?

= when a protein loses active conformation and becomes inactive • Occurs by changing pH, temp, chemicals or even enzymes§ If you denature by heating, you destroy all the structures of the protein except the primary structure (primary structure is conserved)

Base-excision repair

A damaged base gets cut out. Then the base's sugar phosphate backbone gets cut out. Several more nucleotides next to base get cut out. Finally, polymerase remakes the cut-out nucleotides.

What are the hydrophobic amino acids?

Alanine, isoleucine, leucine, methionine, phenylalanine, valine, proline, glycine

Antigen = the ligand for antibodies

Antigens can be thought of as little red flags for the immune system letting us know, "Hey, that's not supposed to be there!" Antibodies are protein components of the adaptive immune system whose main function is to find foreign antigens and target them for destruction

What are the hydrophilic amino acids?

Arginine, lysine, aspartate, glutamate, glutamine, asparagine, histidine, serine, threonine, tyrosine, cysteine, tryptophan

What are the acidic amino acids?

Aspartate and glutamate

How does transcription work?

Chain Initiation: RNA polymerase binds to promoter (TATA box) of dsDNA (closed complex). Double stranded DNA template opens up (open complex) o 2. Chain Elongation: nucleoside triphosphates (AUGCs) adds corresponding to the DNA template. No primer is required. RNA elongates as RNA polymerase moves down DNA template. RNA made from 5' to 3' direction. o 3. Chain Termination: 2 ways that transcription can terminate:§ 1. Intrinsic termination: specific sequences called a termination site creates a stem-loop structure on RNA that causes RNA to slip off template.§ 2. Rho (ρ) dependent termination: a protein called the ρ factor travels along the synthesized RNA and bumps off the polymerase

Lyase

Cleave without the addition of water and without the transfer of electrons (reverse reaction, synthesis, is usually more biologically important.) Lyases generate either a double bond or a ring structure

DNA gyrase

DNA gyrase (class II topoisomerase) responsible for uncoiling the DNA ahead of the replication fork

What kind of proofreading ability does DNA polymerase have?

DNA polymerase has proofreading activity (also called 3'à5' exonuclease activity). If a wrong nucleotide gets incorporated, polymerase will "back-up" and replace it with correct one o Special polymerase that replaces the RNA primers with DNA also have 5'à3' activity. This allows polymerase to clear away short stretches of incorrect nucleotides (RNA or incorrect DNA) and replace it with the right ones (DNA).

What does uncompetitive inhibition do?

Decrease Km and Vmax. It binds the allosteric site. The inhibitor only binds to the enzyme substrate complex

What does a mixed inhibitor do?

Decreases Vmax and can either increase or decrease Km. If it increases Km, then that means that it is binding to the enzyme without the substrate. Or it can decrease Km if it is binding to the enzyme with the substrate already bound

Cofactors

Directly involved in the enzyme's catalytic mechanism (might be stabilizing the substrates, or helping the reaction to convert substrates from one form to another) (e.g. Mg2+)

How does one calculate the turnover number?

Do the Vmax divided by the enzyme concentration

What configuration does every amino acid have? R or S?

EVERY AA has S configuration EXCEPT FOR cysteine (R configuration)

What are the basic amino acids?

Histidine, lysine, and arginine

Nucleotides and nucleosides

Important structural component of DNA which consists of the pentose sugar and phosphate groups. Sugars linked together by a phosphodiester bond

Zymogens

Inactive form of an enzyme that requires covalent modification to become active I.e. Digestive enzymes of the pancreas o Pancreas releases trypsinogen (a zymogen)o Once in the intestine, it is covalently modified by an enzyme called enterokinase to the active form Trypsino This makes sure trypsin does not break down proteins that we need in the pancreas

How does one increase the Vmax?

Increase the amount of enzyme present

What does competitive inhibition do?

Increases Km and no effect on Vmax

Transcription

Inside the nucleus, the DNA genes get transcribed into RNA (messenger RNAs or mRNAs)

Quaternary structure

Interactions between subunits (multiple polypeptides)§ Hydrophobic interactions and ionic bonds between side chains (i.e. cysteine side chains making disulfide bonds)

What does noncompetitive inhibition do?

It decreases Vmax and no effect on the rest. It binds at an allosteric site and decreases the overall rate of the reaction

What does a high Km mean?

It reflects low affinity

Primary structure

Linear sequence of amino acids§ Determined by the peptide bond linking each amino acid's Covalent (Peptide) bonds

Secondary structure

Local structure, stabilized by hydrogen bonding, α-helices - hydrogen bonds run up and down, stabilizing the structure. β-pleated sheets - stabilized by hydrogen bonds connecting the sheets o Antiparallel vs. Parallel configurations. The way the linear sequence folds on itself § Determined by the backbone interactions (primarily hydrogen bonds) § Hydrogen bonds between backbone atoms

Water-soluble vitamins

Need to obtain from the diet, Vitamins = organic cofactors and coenzymes

What charge are amino acids at high pH?

Negatively charged

How does one increase transcription?

Once the transcription complex is formed, basal (or low-level) transcription can begin and maintain moderate, but adequate, levels of the protein encoded by this gene in the cell. There are times, however, when the expression must be increased, or amplified, in response to specific signals such as hormones, growth factors, and other intracellular conditions. Eukaryotic cells accomplish this through enhancers and gene duplication

Gene repression in bacteria

Operons have a binding site for regulatory proteins that turn expression of the operon "up" or "down." Some regulatory proteins are repressors that bind to pieces of DNA called operators. When bound to its operator, a repressor reduces transcription (e.g., by blocking RNA polymerase from moving forward on DNA)

Coenzymes

Organic carrier molecules (i.e. NADH, CoA)

What charge are amino acids at low pH?

Positively charged

Electrophoresis

Positively charged anode at bottom, negatively charged cathode at top. Larger molecules will have harder time moving, thus separation created by size with the smallest molecules towards the bottom

What is a ribozyme?

RNA molecule that is capable of catalyzing specific chemical reactions

snRNPs

RNA-protein complexes that combine with unmodified pre-mRNA and various other proteins to form a spliceosome, a large RNA-protein molecular complex upon which splicing of pre-mRNA occurs

DNA

Resides in nucleus. Codes information in genes.

Positive control in bacteria

Some regulatory proteins are activators. When an activator is bound to its DNA binding site, it increases transcription of the operon (e.g., by helping RNA polymerase bind to the promoter)

DNA synthesis

Synthesis that proceeds in direction of replication fork is leading strand. Synthesis that proceeds in opposite direction to replication fork is lagging strand. Lagging strand contains Okazaki fragments.

Absolute configuration at the α position?

The alpha carbon IN EVERY amino acid is a chiral center EXCEPT in glycine (it is achiral, since the R group is an H)

Why is protein folding favorable?

The hydrophobic regions of the protein aggregate, which releases the water from cages àThis increases the entropy of water, which is the major thermodynamically favorable component of protein folding

RNA

The mRNAs get transported out of nucleus into cytoplasm. mRNAs are working copies of the gene.

Silencers

The main difference in eukaryotes from prokaryotes is that enhancers/silencers can be very far away from actual promoter and can be upstream or downstream. The DNA must loop back on itself so that the transcription factor bound to enhancer/silencer can make contact with promoter. Intermediate proteins are involved in the process.

How is a peptide bond formed?

The nucleophilic amino group attacking an electrophilic carbonyl. The bond when formed has a lot of resonance delocalization (partial double bond character all over the place!) However, this is still free rotation around the ALPHA CARBON

What does it mean to be a zwitterion?

The pH = pI

Hydrolysis

The process of breaking the peptide bond • Done by either acid/base hydrolysis (nonspecific) or with the help of proteolytic enzymes (specific)

What is the Michaelis-Menten equation?

V= Vmax(s)/Km + S

Activator

When an activator is bound to its DNA binding site, it increases transcription of the operon (e.g., by helping RNA polymerase bind to the promoter)

Operon

a cluster of genes transcribed as a single mRNA. The numerous genes share a single common promoter region on the DNA sequence and are transcribed as a group.

Centromere

a region on the chromosome, can be at the center or close to one of the ends. After replication, sister chromatids are attached at the centromere. During mitosis, spindle fibers are attached at the centromere and pulls the sister chromatids apart.

Cysteine

amino acid with the thiol R group

Nick translation

basically 5'à3' exonuclease activity coupled to polymerase activity. The polymerase chugs along, chews off bad nucleotides and replaces them with new nucleotides. This is what happens when RNA primers are replaced with DNA.

SDS-PAGE

break into subunits

Telomerase

catalyzes lengthening of telomeres; enzyme includes molecule of RNA that serves as template for new telomere segments

Electrostatic catalysis

charged molecules or metal ions used to stabilize big positive or negative charges

Structural gene

codes for protein of interest

Regulator gene

codes for the repressor protein

snRNAs

complexed with proteins to form snRNPs to splice primary RNA transcripts.

Nucleotide-excision repair

damaged nucleotide(s) get cut out then polymerase replaces it. This is like mismatch-repair, but not for mismatch. It's for damages like thymine dimers, and other damages that changes normal nucleotides into abnormal nucleotides.

Alternative splicing

different ways of cutting up the RNA and rejoining the exons pieces makes different final RNA products

SOS response in E. Coli

during replication, when there's too much DNA damage for normal repair to handle, the SOS repair system comes along. Instead of correcting any DNA damages during replication, polymerase replicates over the damaged DNA as if it were normal. By using damaged DNA as template error rates are high, but still better than not replicated at all.

Covalent catalysis

enzymes covalently bind to help with electron transfer

Proximity/Orientation effects

enzymes make collisions between reacting molecules happen more often

Mismatch repair

enzymes recognize incorrectly paired base-pairs and cuts out stretch of DNA containing the mismatch. Then polymerase re-adds the correct nucleotides in. During mismatch repair, repair enzyme must decide what strand of DNA to cut since DNA contains 2 strands. To do this, the enzyme cuts DNA strand that does not have methylations. The original (old) DNA has methylations but newly synthesized DNA does not have them until shortly after replication. Thus, there is a short period when mismatch repair enzymes can find out what strand to cut if mismatch is encountered.

Acid/Base catalysis

enzymes use acidic/basic properties to make rxns go faster by proton transfer

What does dispersive replication mean?

everything would be of intermediate weight. This was not the case because after second round of replication, light DNA was seen.

Nuclease

excises or cuts out unwanted or defective segments of nucleotides in DNA sequence

Isoelectric focusing

gel electrophoresis method that separates proteins on basis of their relative contents of acidic and basic residues (gel with pH gradient is used)

Ligase

glues together Okazaki fragments, an area DNA Pol I unable to synthesize

Eukaryotes

have multiple origins of replication across their numerous linear chromosomes

Single Strand Binding Proteins

holds the replication fork of DNA open while polymerases read the templates and prepare for synthesis

How does gene duplication help?

increase expression of a gene product by duplicating the relevant gene.

Enhancers

increase transcription when bound

Topoisomerase

introduced single-strand nick in the DNA, enabling it to swivel and thereby relieve the accumulated winding strain generated during unwinding of double helix

Primase

lays down short RNA primer on unwound DNA. Primer made of RNA but is complementary to DNA sequence. Later, this RNA is replaced with DNA.

What do kinesin and dynein do?

motor proteins responsible for intracellular transport

Operator site

nontranscribable region of DNA capable of binding a repressor protein

What does post-transcriptional modification do?

o Post-transcriptional modifications include addition of the 5' cap, polyA tail and splicingo In prokaryotes, mRNAs with better Shine-Dalgarno sequence are translated moreo In eukaryotes, post-transcription regulation can involve adding more polyAs to mRNA (longer mRNA life time), modulation of the translation machinery (phosphorylation of initiation factors), or storing mRNAs to be translated at a later time (mRNA masking)o The cap and polyA tail are added co-transcriptionally, but still considered post transcriptional o Splicing gives rise to isoforms. Depending on how you arrange the introns/exons, you get different proteins by alternative splicing.

Suicide inhibition

o Suicide inhibitors covalently bind the enzyme and prevent it from catalyzing reactionso Rarely unbind - why it's called suicide (enzyme won't work anymore)

What does conservative replication mean?

only heavy and light DNA would be seen, nothing in between.

What is the isoelectric point?

pI is determined by averaging the pKa values that refer to the protonation and deprotonation of the zwitterion

Primase

polymerizes nucleotide triphosphates in a 5' to 3' direction. Synthesizes RNA primers to act as a template for future Okazaki fragments to build on to.

Antibodies

protein components of the adaptive immune system whose main function is to find foreign antigens and target them for destruction

Promoter site

provides a place for RNA polymerase to bind.

Single-strand binding protein

responsible for keeping DNA unwound after helicase. SSBs stabilize ssDNA by binding to it.

Helicase

responsible for unwinding DNA at replication fork

What does PAGE do?

retains structure of protein

RNA splicing

sequences called introns cut out, sequences called exons are kept and spliced (joined) together

Prokaryotes

single origin of replication for their single, circular DNA

DNA Polymerase III

synthesizes nucleotides onto leading end in classic 5' to 3' direction.

DNA Polymerase I

synthesizes nucleotides onto primers on lagging strand, forming Okazaki fragments. This enzyme cannot completely synthesize all the nucleotides.

DNA polymerase

takes over and makes DNA that is complementary to unwound DNA.

DNA polymerase

takes over and makes DNA that is complementary to unwound DNA. DNA synthesis occurs on both strands of unwound DNA. Synthesis that proceeds in direction of replication fork is leading strand. Synthesis that proceeds in opposite direction to replication fork is lagging strand. Lagging strand contains Okazaki fragments.

5' cap and poly-A tail

these help to protect the RNA from degradation, so they can last longer

Helicase

uses hydrolysis of ATP to "unzip" or unwind DNA helix at replication fork to allow resulting single strands to be copied

Supercoiling

wrapping of DNA on itself as its helical structure is pushed even further toward the telomeres during replication. To alleviate the torsional stress and reduce risk of strand breakage, DNA gyrase (DNA topoisomerase II) introduces negative supercoils.