Translation and Protein Synthesis

Question 0

Mutations

Answer 0

Insertion or deletion of a base disrupts reading frame
Stop codon gives truncated protein
Point mutations are silent mutations if no amino acid change, but can still affect rate of translation and polypeptide folding, end with proteins with different properties

Question 1

Genetic code

Answer 1

4^n where n=number of bases per amino acid
64 possible code words for 3 bases
Code words are non overlapping
Genetic code universal except in mitochondria
Is degenerate: multiple codons encode a single amino acid
Start codon (AUG) encodes Met and establishes reading frame
Three stop codons (UAA, UAG, UGA)

Question 2

Transfer RNA

Answer 2

Cloverleaf maintained by hydrogen bonding
Base pairing between 5’ and 3’ ends form acceptor or amino acid stem, amino acid attached at 3’ by specific amino-acyl synthetase to make amino acyl-tRNA
Anticodon loop interacts with codon of mRNA

Question 3

Wobble hypothesis

Answer 3

No tRNA for every codon

First two bases of codon:anticodon interaction are constrained but third base H bond is less strict

Question 4

Aminoacyl-tRNA synthetase reaction occurs in 2 steps

Answer 4

1) activation of amino acid by reaction with ATP to form amino acyl adenylate (hydrolysis of pyrophosphate to inorganic phosphate drives reaction forward)
2) reaction of activated amino acid with 3’-OH of tRNA to form amino acyl-tRNA

2 high energy phosphoanhydride bonds hydrolyzed for each amino acid

Question 5

Ribosomes

Answer 5

Ribosomal RNA (ribozyme) and protein
80S ribosome with 60S large subunit and 40S small subunit
Activity at interface between large and small subunits
Large subunit has aminoacyl site, peptidyl site, exit site

Question 6

Initiation in prokaryotes

Answer 6

Shine-Dalgarno sequence for recognition of reading frame, upstream of start codon
Polycistronic messages (multiple proteins can be translated fro, same mRNA) due to alignment of ribozyme at different shine-Dalgarno sequences
Used formyl methionine-tRNA
Three initiation factors: IF1 (assists IF3), IF2 (binds to fMet-tRNA and GTP, allow binding to small subunit), IF3 (binds small subunit to promote dissociation of subunits and help recognize shine-Dalgarno sequence

Question 7

Initiation in eukaryotes

Answer 7

7-methyl guanosine residue (5’ cap) allows recognition of mRNA by ribosomes
Monocistronic (single protein translated from mRNA )
Initiator is Met
Have more than 10 initiation factors: eIF-2 is equal to IF-2
GTP accelerates reactions
Complex of eIF-2 + GTP + Met-tRNA along with mRNA bind to small subunit
Hydrolysis of GTP allows large subunit to bind as initiation factors are expelled
Initiator tRNA is moved to the P site, first peptide bond is formed, move initiator tRNA to E site

Question 8

Elongation factors

Answer 8

Prokaryotes:
EF-Tu binds to GTP and aminoacyl-tRNA, brings them to A site, required hydrolysis of GTP, end with inactive EF-Tu-GDP complex
EF-Ts regenerates EF-Tu
EF-G moves mRNA from A site to P site, requires GTP hydrolysis
Eukaryotes:
eEF-1 has two subunits that perform functions of EF-Tu and EF-Ts
eEF-2 is equal to EF-G

Question 9

Elongation

Answer 9

EF-Tu forms complex with GTP and aminoacyl-tRNA
Complex binds to ribosome at A site, kicks out tRNA in E site
GTP hydrolyzed, EF-Tu + GDP dissociates
Transpeptidation: formation of peptide bond, transfer of peptide chain to tRNA in A site by peptidyl transferase activity in large subunit, no energy required because aminoacyl tRNA is charged
Translocation: uncharged tRNA left in P-site moves to E site, peptidyl tRNA translocated from A to P, requires GTP hydrolysis

Question 10

Termination

Answer 10

Stop codons are recognized by release factors with GTP
Binding of release factor alters activity of peptidyl transferase, add H2O instead of amino acid to peptidyl-tRNA, uncharged tRNA to E site
GTP hydrolyzed so mRNA, tRNA, RFs and GDP + P are released

Question 11

Sources of error

Answer 11

Attachment of wrong amino acid to tRNA

Incorrect base pairing of tRNA to the codon

Question 12

Proof reading

Answer 12

Amino acyl synthethases have two active sites (one recognizes correct tRNA and attaches the amino acid and other can recognize and remove an incorrectly attached amino acid, costs 2 phosphoanhydride bonds)

When aminoacyl-tRNA first binds to A site, EF-Tu:GDP complex remains associated (if base pairing correct then released and peptide bond forms, if not correct, aminoacyl-tRNA dissociated along with EF-Tu:GDP

Question 13

Regulation of translation: phosphorylation of eIF-2

Answer 13

eIF-2-GTP brings initiator tRNA to small subunit, inactivated as GTP hydrolyzed and initiation complex formed
Normally eIF-2-GDP is recycled to eIF-2-GTP via interaction with eIF-2B
But phosphorylation of eIF-2-GDP by protein kinase locks eIF-2/eIF-2B complex in inactive, GDP-bound form

Question 14

Regulation of synthesis of globin in response to heme availability

Answer 14

Globin synthesized only when heme is available
In absence of heme, cells activate HCI (heme-controlled inhibitor) which phosphorylates eIF-2.
HCI inactivated when heme available, phosphate removed, eIF-2 can be recycled

Question 15

Regulation of translation:

Regulatory elements within structure of eukaryotic mRNA

Answer 15

5’ cap protects end from ribonucleases, allow cells to distinguish between mRNA and other RNAs, recognized by eIF and is required for translation
5’ untranslated region contain sequences important for translational efficiency
3’ untranslated region contain signal sequences that target mRNA to be translated at specific places or to be transported to particular location to concentrate, also for mRNA stability
Poly A tail stabilizes 3’ end of mRNA, catalyze assembly of large subunit

Question 16

Regulation of ferritin/transferrin receptor translation in response to iron availability

Answer 16

Translation of mRNA for Ferritin (intracellular iron storage protein) is increased and translation of mRNA for transferrin receptor (imports iron) decreases if [iron] increases
If [iron] low: binding of aconitase (iron response factor) to IRE (iron response element) in 5’UTR of ferritin mRNA blocks initiation of translation, binding of aconitase to IRE in 3’UTR of transferrin receptor mRNA stabilizes against degradation

Question 17

Regulation of translation:

Regulation of protein synthesis by micro and small interfering RNA molecules (miRNA, siRNA)

Answer 17

Endogenous miRNA and siRNA can down regulate translation by inducing mRNA degradation
Exogenous dsRNA gets processed to siRNA

Question 18

Antibiotics

Answer 18

Act to inhibit different phases of protein synthesis

Question 19

Interferons

Answer 19

DsRNA not normally present in cells
if present, then viral infection, induce secretion of interferons (IFNs)
IFNs bind surface of other cells, induce expression of 2 enzymes that when activated by presence of dsRNA inhibits protein synthesis
-ribosome-associated protein kinase phosphorylates eIF-2 and prevents initiation of translation
-2,5A synthetase produces unusual polymers of ATP that activate endoribonuclease that cuts in middle of both mRNAs and rRNAs, slows down protein synthesis