Translation (8-9)

Question 1

What is translation?

Answer 1

The joining of aminoacyl residues by the ribosome to form a polypeptide
→ encoded by triplets
→ high energy cost to cell - essential that its only carried out when required

Question 2

What are the components of the ribosome?

Answer 2

Prokaryotic → 50S + 30S = 70S

Eukaryotic → 60S + 40S = 80S

→ overall architecture similar between the types

Question 3

Are secondary structures of ribosomes conserved?

Answer 3

rRNAs of E. coli first sequenced in 1978

rRNAs from several hundred species have been sequenced
→ all show some defined structures

Conserved regions of rRNA → mutations in these regions are often fatal

Variable regions → can tolerate mutations

Base-paired stems (alpha helix) common
→ compensating base changes between species give the same overall structure - allows for natural folding that the ribosome wants to take

Question 4

How are ribosomes structured?

Answer 4

When the subunits come together - forms 3 pockets
→ 3 binding sites for tRNA that span the 30S and 50S subunits

A = acceptor site - of codon-directed binding of incoming aa tRNA
P = peptidyl site - holds codon directed peptidyl tRNA (formation of new peptide)
E = exit site - not associated with mRNA (release)

Question 5

What is the role of 23S rRNA in the peptidyl transferase of translation?

Answer 5

Catalyses the formation of peptide bonds between amino acids
→ RNA driving the reaction - ribozyme activity

N3 accepts a proton from the amino group of the aminoacyl tRNA in the A site
→ enhances the negative charge of the amino group allowing it to attack the bond between the peptide and tRNA in the P site
→ the N3 H-bonds to the oxyanion in the tetrahedral intermediate stabilising it and accelerating the reaction

The 3’-OH of the tRNA in the P site accepts the proton completing the reaction

Question 6

Are the proteins of the peptidyl transferase active site in the 50S subunit P site involved in catalysis?

Answer 6

The nearest protein to the active site is 18.4 Angstroms from the active site
→ therefore is too distant to participate in catalysis

Question 7

What is the poly-peptide exit tunnel in the 50S subunit?

Answer 7

Exit tunnel for peptide chain to leave
→ shape, size and hydrophobic residues - slippy
→ allows for protein folding - alpha helical proteins come out already

Question 8

What are isoaccepting tRNAs?

Answer 8

Several different tRNAs (often with different anticodon sequences) can become charged with the same amino acids
→ ester bond between aa and tRNA

Question 9

What are the aminoacyl-tRNA synthetases?

Answer 9

Family of enzymes which charge tRNAs with their cognate aa
→ show specificities for the tRNAs they charge
→ very rarely is a non-cognate tRNA is aminoacylated
→ forms an ester bond between tRNA and aa
→ each synthetase recognises a single aa and all the tRNAs that should be charged by it

Question 10

What is involved in the cloverleaf model for tRNA?

Answer 10

D loop → contains 8-12 unpaired bases, and 2-3 dihydrouricil residues

Anticodon loop → 7 unpaired bases
→ contains 3 anticodon bases, anticodon flanked by U on the 5’ side and an alkylate purine on the 5’ side

Variable loop → varies in size

T loop → 7 unpaired bases, involved in binding to the ribosome ‘A’ site

3’ end → unpaired bases (CCA), A - amino acid attachment

Paired sections → STEMS between loops - gives structure
→ closely controlled sites - provide 3D structure

Question 11

How is yeast tRNA structured?

Answer 11

Tertiary structure → more L shaped, not clover, interaction between the T and D loops
→ the 3 anticodon bases and the -CCA-3’ bases are unstacked - allowing interaction with the codon base or the aminoacyl-tRNA synthetase
→ many of the tertiary H-bonding interaction involve bases that are invariant in all known tRNAs - supporting the belief that all tRNAs have basically the same structure

Question 12

What are the shared and unique reactions of all tRNAs?

Answer 12

Shared reactions of all tRNAs
→ interaction with elongation factor (except initiator tRNA)
→ binding to the ribosome ‘A’ site
→ CCA terminal addition - allows aa to bind
→ invariant modifications to bases

Unique reactions of individual tRNAs
→ amino acylation by synthetases
→ codon-anticodon interaction
→ recognition of initiator (feet tRNA) by initiation factor
→ recognition of initiator by transformylase
→ unique base modifications

Question 13

How are tRNAs charged by aminoacyl-tRNA synthetases?

Answer 13

1. A specific aa and ATP bind to the aminoacyl-tRNA synthetase
2. The aa is activated by the covalent binding of AMP
   → pyrophosphate is released
3. The correct tRNA binds to the synthetase
   → the aa is covalently attached to the tRNA, AMP is released
4. The charged tRNA is released

Question 14

What are the classes of tRNA synthetases?

Answer 14

Class I → contacts tRNA at minor groove of the acceptor stem and anticodon
→ alternating alpha helix and beta sheets

Class II → contacts tRNA at major groove of the acceptor stem and anticodon
→ core of beta sheets surrounded by alpha helices

The classes recognise different faces of the tRNA molecule → the CCA arm adopts different conformations with the two classes
→ the way they interact looks different - different folding and binding - but they do the same job to their cognate tRBA

Question 15

What are the features of individual tRNAs which are recognised by their cognate synthetase?

Answer 15

Identity elements → essential for the accuracy of protein synthesis
→ more interactions than just at the anticodon and acceptor ends - also in bends of the L loop

Question 16

How are aminoacyl-tRNA synthetases involved in proof reading?

Answer 16

Two stages (double sieve)

1. Hydrolysis of the ester bond of an ‘incorrect’ aminoacyl-AMP intermediate triggered by the binding of the cognate tRNA
2. By hydrolysis of the ester bond of a ‘miss-matched’ aminoacyl-tRNA

→ if aa too big won’t fit, if too small won’t be held firmly
→ the flexible CCA arm of an aminoacyl-tRNA can move the aa from the activation site to the editing site - if the aa fits well into the editing site its removed by hydrolysis

Question 17

How does streptomycin work?

Answer 17

Binds to the 16S rRNA of the 30S subunit of the bacterial ribosome
→ interferes with the binding of formylmethionyl-tRNA to ribosomes
→ prevents correct initiation of protein synthesis

Question 18

How does puromycin work?

Answer 18

Puromycin resembles the amino acyl part of aminoacyl tRNA
→ enters the vacant A site without the involvement of EF-Tu
→ it is a substrate for peptidyl transferase through its amino group forming peptidyl puromycin - not anchored to the A site, dissociates
→ results in premature chain termination

Question 19

How does diphtheria toxin work?

Answer 19

Produced by pathogen strains of Coryebacterium diphtheriae - highly toxic, pulled in as a vesicle
→ it acts catalytically on elongation factor 2 (EF-2) - the eukaryotic homologue of EF-G
→ all EF-2s contain a postranlationally modified histidine residue called dipthamide - the toxin transfers ADP ribose from NAD+ to the imidazole ring
→ completely inhibits translocation

Question 20

How do the initiation and termination regions differ in eukaryotic and prokaryotic mRNA?

Answer 20

Eukaryotic mRNA (monocistronic) → 5’ cap, AUG start codon, ORF, UAA/UAG/UGA stop codon, poly(A) tail
Prokaryotic mRNA (polycistronic) → 5’ ribosome-binding site (Shine-Dalgarno sequence), AUG/GUG start codon, ORF1, UAA/UAG/UGA stop codon
→ repeats for multiple ORF, no poly(A) tail

Question 21

What is the start sequence for prokaryotic translation?

Answer 21

Start signal is AUG (or GUG) - pairs with initiator tRNA
→ upstream from this is a purine-rich region (Shine-Dalgarno sequence) complementary to the initiator sites of mRNA - pairs with 16s rRNA

Question 22

What are the two types of tRNA^met that E. coli uses during prokaryotic translation?

Answer 22

tRNAf^met → met residues attached are formylated
→ initiates polypeptide chains only, recognises AUG and GUG
→ can only be used during process of initiation

tRNAm^met → met residues are only attached, not formylated
→ recognises codon AUG only, used as a source of internal met residues
→ can only be used during process of expansion - not recognised by initiation factors

Question 23

How was evidence of protein ‘factors’ in prokaryotic initiation found?

Answer 23

Initiation of protein synthesis in bacteria require free 30s subunits
If the 30s subunits are washed in high salt (dissociates the proteins) the subunits lose their ability to initiate protein synthesis
→ just ribosomal protein present - not enough to initiate protein synthesis
If the supernatant was dialysed (to remove the salt) and added back - activity restored
→ chromatography of the high salt supernatant revealed 3 proteins necessary for initiation - IF-1, IF-2 and IF-3

Question 24

What is in the 30S initiation complex for translation initiation in prokaryotes?

Answer 24

IF-3: 22kDa → binds the 30S subunit and prevents association with the 50S subunit
IF-1: 9kDa → binds near the A-site - directs fmet-tRNA to the P site
IF-2: 100kDa → reacts with fmet-tRNA and GTP to form a ternary complex IF-2-GTP-fmet-tRNA
→ delivers the ternary complex and mRNA to the partial P site in the 30S subunit-mRNA complex
→ triggers GTP hydrolysis when the 50S subunit joins the complex
→ does not recognise met-tRNA or any aa tRNA used for elongation

Question 25

What is in the 70S initiation complex for translation initiation in prokaryotes?

Answer 25

Dissociation of the 3 IFs from 30S subunit → large 50S can bind, forming the 70S initiation complex → GTP is hydrolysed - priming ribosome uses energy Result: primed ribosome with first methionine sat in the pocket → ready to do a peptidyl transferase on the next incoming tRNA - to start process of elongation

Question 26

What are the 3 steps of elongation in protein synthesis?

Answer 26

1. Codon-directed binding of the incoming aminoacyl-tRNA - with the next aa attached 2. Peptide bond formation → between existing polypeptide and incoming tRNA 3. Translation of the ribosome along the mRNA in a 5' to 3' direction by the length of one codon (3 bases) → forming polypeptide chain

Question 27

How is the peptide bond formed during prokaryotic translation?

Answer 27

The aminoacyl portion of fMet-tRNA is transferred to the amino acid group of the aa residue in the A site - forming a peptide bond → peptidyl transferase activity due to the ribosome function of 23S rRNA → tRNA have high energy bond holding the aa thus energy required to carry out the activity in the ribosome is effectively 0 (energy needed is stored in the bonds)

Question 28

What is the overall mechanism of translocation in elongation during prokaryotic translation?

Answer 28

1. Elongation factors attached to tRNA → moves into vacant A site (orde of sites: EPA) 2. Proofreading → makes sure right aa attached to correct tRNA, incorrect base-paired tRNAs preferentially dissociate 3. GTP hydrolysis allows for a reorganisation of the tRNA → can come into close contact with tRNA in P-site - allows peptidyl transferase to occur 4. Chain transfers to A site 5. EF-G slots into partial A site → EF-G/GTP binds the pre-translocation ribosome at a site including L7/L12, L11 and the sarin/ricin loop of 23S rRNA 6. GTP hydrolysis → induces conformational change in EF-G forcing its arm deeper into the 30S subunit - moves peptidyl tRNA from A to P site carrying the mRNA and deacylated tRNA with it Result: empty A site, tRNA in E site ready to be kicked out, ribosome moved along by one codon

Question 29

What is the function of the prokaryotic elongation factors?

Answer 29

EF-Tu → brings in each aa-tRNA, most aa-tRNAs are complexed with EF-Tu → does not react with met-tRNA^met - why this is not bound during elongation → protects the ester bond between tRNA and aminoacyl residue from hydrolysis keeping them attached - means only peptidyl transferase reaction occurs in pocket → incoming tRNA with EF-Tu has distortion - holds active site away until proof reading done - EF-Tu removed allows active sites to be close together EF-G → facilitates translation to next codon → switch protein that switches when you hydrolyse ATP - pulls ribosome along by one codon EF-Tu: RNA and protein, EF-G: only protein, both have very similar shape, N-termianl region of EF-G mimics tRNA → allows EF-G to fit into the pockets and do job of affecting translocation process

Question 30

What is EF-Tu's proof-reading role in translation?

Answer 30

When you've got incoming tRNA with EF-Tu attached there's distortion → bent away to allow for proofreading Only after EF-Tu-GDP release can peptide bond formation occurs (takes a few milliseconds for GTP hydrolysis and EF-Tu release) → the short breaks provide opportunity for weakly bound, non-cognate aa-tRNA to dissociate from ribosome

Question 31

What is involved in prokaryotic translation termination?

Answer 31

1. At the stop codon (don't want more aa) → 3 release factors bind: RF1 (UAA+UGA), RF2 (UAA+UAG), RF3-GTP (aids binding) 2. RF binds vacant A site 3. Peptidyl transfer of the peptidyl group to water - rather than an aminoacyl tRNA 4. Hydrolysis of RF3-GTP to GDP dissociates everything → not attached to A or P site

Question 32

What are the roles of translational initiation factors in prokaryotes / eukaryotes?

Answer 32

Prokaryotes: IF-1 → prevents premature binding of tRNAs to the A site IF-2 → guides fMet-tRNA^met to the 30S subunit IF-3 → prevents premature association of 50S subunit Eukaryotes: elF1 → guides met-tRNAi to the 40S subunit elF2B, elF3 → first binders facilitating later steps elF4A → RNA helices that unwinds secondary structures elF4B → binds the mRNA and facilitates scanning elF4E → binds the 5'-cap of mRNA elF4G → binds elF4E and pol(A) binding protein, circularising the mRNA elF5 → promotes dissociation of initiation factors elF6 → promotes dissociation of 80S into 40S and 60S subunits → binding to cap then scanning for start codon - requires more factors

Question 33

What is involved in initiation of protein synthesis in eukaryotes?

Answer 33

AUG almost always used as the initiation codon A special initiator tRNA, tRNAi^met (mot formylated), is used as the initiator → tRNAm^met is used to insert internal methionines The 'first' AUG is usually used for ignition (~90%) → context dependant, preferred orientation: KOZAK sequence → there is no ribosome binding site as in prokaryotes

Question 34

What is in the cap binding complex (elF-4F) in eukaryotes?

Answer 34

elF4E → binds 5' cap elF-4A → ATP-dependent RNA helices that removes secondary structures - needed for scanning movement of 40S subunit along mRNA elF-4G → 'scaffold' subunit - links together the initiation complex, cleavage by protease results in inhibition of cap initiation → interacts with poly(A) binding protein - allows for pseudo circularisation of mRNA

Question 35

How does elF4G increase efficiency of translation?

Answer 35

Poly(A) binding protein interacts with elF4G and elF4E bound to cap - pseudo circularisation of mRNA → stabilises mRNA → ribosomes that have completed translation dissociate into subunits - can readily find the nearby m7G gap and initiate another round of protein synthesis - increasing efficiency

Question 36

What are internal ribosome entry sites (IRES)?

Answer 36

The vast majority of eukaryotic mRNAs are translated through ribosome scanning mechanism - IRED is alternative mechanism → the mRNAs lack a 5' cap → IRESs have a complicates tertiary structure and bind 40S subunits in close proximity to an AUG codon → still get circularisation of mRNA During mitosis cap-binding translation reduced → caused by dephosphorylation of 4E → relative amount of IRES translation increases → picornaviruses clips 4G so can't associate with cap - less cap translation, left with just IRED - gives advantage over host

Question 37

How are eukaryotic release factors structured?

Answer 37

Mimics the structure of the aa acceptor stem of tRNA (CCA terminus) → the sequence Gly Gly Gln at the tip of the acceptor stem binds a water molecule which is carried into the peptidyl transferase centre of the ribosome → this water hydrolyses the ester bond of peptidyl tRNA releasing the polypeptide → during chain elongation water is excluded from the peptidyl transferase centre of the ribosome - don't usually have water in ribosome pockets to prevent early termination

Question 38

What are some possible translational control mechanisms?

Answer 38

→ regulation of the activities of initiation and/or elongation factors by phosphorylation → blocking/opening of ribosome binding sites by reversible changes in secondary structure (prokaryotes) → autogenous regulation - protein product of a gene binds to ribosome binding site in mRNA preventing initiation (prokaryotes) → reversible binding of a repressor protein to a response element in 5' UTR (eukaryotes) → differential stability of mRNA

Question 39

How can elF2a phosphorylation control initiation?

Answer 39

Signal transduction from cell → viral infection - PKR, heme deprivation, oxidaitve stress, heat shock - HRI, ER stress - PEK, nutrient limitation, proteasome inhibition, UV irradiation - GCN2 Cause phosphorylation of elF2 → blocks exchange factor (elF2B) to recharge elF2 with GTP - blocking translation initiation

Question 40

How does *E. coli* use autogenous control of ribosomal protein synthesis?

Answer 40

The demand for r-proteins is growth rate dependant and closely couples to rRNA synthesis → r-protein synthesis is regulated at the level of translation → when rRNA is in short supply levels of free r-proteins increase → one r-protein from each operon then binds to the polycistronic mRNA near to the ribosome binding site - prevents translation of this and other ORFs → autogenous (self limiting) control

Question 41

How is the translation of mammalian ferritin and transferrin mRNAs regulated by iron-response element binding proteins?

Answer 41

Ferritin is a cytosolic protein that binds iron ions and prevents accumulation of toxic levels → when Fe is limiting ferritin poses a problem - competes for Fe with iron-requiring enzymes → thus mammalian cells modulate synthesis of ferritin → the transferrin receptor shows reciprocal regulation of synthesis to that of ferritin → aconitase undergoes conformational change on iron binding: IRE released ferritin mRNA is translated, transferrin receptor mRNA is degraded Iron starvation - no ferritin made, excess iron - ferritin made

Question 42

What is involved in eukaryotic mRNA decay?

Answer 42

Nearly all mRNAs subjected to poly(A) tail shortening → when tail <30 As - poly(A) binding protein lost and 3' end no longer associates with cap → leads to decapping followed by degradation Cleavage at the endonuclease cleave site in the 3' UTR → decapping followed by degradation