Lecture 9. The Mechanics of Translation

Question 1

What are the key differences between eukaryotic and prokaryotic mRNA?

Answer 1

Have different initiation and termination regions
Eukaryotic mRNA has a 5’ cap ahead of the start codon, prokaryotic mRNA has a ribosome binding site (RBS)
Eukaryotic mRNA has a poly(A) tail at the 3’ end, prokaryotic mRNA does not

Question 2

What is the start signal in prokaryote mRNA?

Answer 2

AUG (or sometimes GUG) preceded by several bases that pair with 16S rRNA
The purine-rich region, called the Shine-Dalgarno sequence is complementary to the
initiator sites of mRNA

Question 3

What are the two types of tRNAmet (targets methionine/Met) in E. coli?

Answer 3

tRNAfmet : Met residues attached to this are formylated
Initiate polypeptide chains only
Recognises AUG and GUG (GUG internally codes for valine)
Can only be used in the initiation of translation

tRNAmmet : Met residues are only attached, not formylated
Recognises the codon AUG only
Used as a source of internal met residues
Can only be sued during the process of extension

Question 4

In the prokaryotic system, what is required for protein synthesis?

Answer 4

Initiation of protein synthesis in bacteria require free 30S subunits and three proteins (factors) that are necessary for initiation. Termed IF-1, IF-2 and IF-3

Question 5

How is translation initiated in the 30S subunit of prokaryotic ribosomes?

Answer 5

IF-3: binds to the 30S subunit and prevents association with the 50S subunit
IF-1 binds near the A site therefore directs fmet-tRNA to the P site
IF-2 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
It does not recognise met-tRNA or any amino acid (aa) tRNA used for elongation

Question 6

How is translation initiated in the 70S subunit of prokaryotic ribosomes?

Answer 6

The 50S subunit joins the 30S initiation complex to form the 70S initiation complex
Initiation factors are released and GTP is hydrolysed

Question 7

What happens in elongation?

Answer 7

Codon-directed binding of the incoming aminoacyl-tRNA
Peptide bond formation
Translocation (movement) of the ribosome along the mRNA in a 5’ → 3’ direction by the length of one codon (move 3 bases)

Question 8

How is a peptide bond formed by peptidyl transferase?

Answer 8

The aminoacyl portion of fMet-tRNA is transferred to the amino acid group of
the amino acid (aa) residue in the A site, forming a peptide bond
Activity due to the ribozyme function of 23S
Overall Keq ~1. therefore no energy input is required

Question 9

Overview o the mechanism of translocation

Answer 9

Elongation factors attached to incoming amino acid (aa) tRNA moves into vacant A site
Proof reading step to make sure right aa is attached to the right tRNA
GTP hydrolysis allows for reorganisation of tRNA, allowing it to come into contact with the tRNA in the P site, allowing peptide transferase to occur
Peptide chain transfers to A site tRNA
EF-G facilitates translocation of ribosome along the mRNA
EF-G slots into partial A site, hydrolysis of GTP forces the EF-G into the rest of the A site and pulls the ribosome along.
Peptide now in the P site, A site now empty again, previous tRNA now in exit site

Question 10

What is the role of the elongation factors EF-Tu and EF-G?

Answer 10

EF-Tu brings in each aa-tRNA
EF-G facilitates translocation to next codon

Question 11

What does the binding of the incoming aminoacyl-tRNA require?

Answer 11

A soluble supernatant factor, elongation factor T (EF-T) and GTP

Question 12

What are the two polypeptides of EF-T?

Answer 12

EF-Tu, 45kDa (heat unstable)
EF=Ts, 30kDa (heat stale)

Question 13

What are the characteristics of EF-Tu?

Answer 13

EF-Tu is very abundant (~20 mols per ribosome). Most aa-tRNAs in the cell are complexed with EF-Tu
EF-Tu does not react with fmet-tRNAmet explaining why this is not bound during elongation
When bound to EF-Tu the labile ester bond between the tRNA and aminoacyl residue is protected from hydrolysis

Question 14

How does EF-Tu interact with the Active (A) site of the ribosome?

Answer 14

Bends the A site to hold active site away until proofreading has happened and check the right aa, tRNA and the right codon are all matching
EF-Tu released from A by GTP hydrolysis, A site returns to original position

Question 15

What is EF-Tu proofreading role in translation?

Answer 15

It takes a few milliseconds for GTP hydrolysis to occur, and a few more milliseconds for EF-Tu-GDP release. Only after EF-TU-GDP release can peptide bond formation occur. These intervals provide the opportunity for a weakly bound, non-cognate aa-tRNA (incorrect codon-anticodon match) to dissociate from the ribosome

Question 16

What is the movement of the ribosome along the mRNA in translocation effected by?

Answer 16

Elongation factor G/GTP (EF-G/GTP) in bacteria or EF-2 in eukaryotes

Question 17

What is EF-G structurally similar to?

Answer 17

The structure of EF-G is very similar to the EF-Tu
The structure of the N-terminal region of EF-G mimics the tRNA

Question 18

What is the mechanism of EF-G/GTP in translocation?

Answer 18

EF-G/GTP binds to the pre-translocation ribosome at a site including L7/L12, L11 and the sarcin/ricin loop of 23S rRNA
The tRNA-like domain interacts with the 30S subunit close to the partial A site
GTP hydrolysis induces a conformational change in EF-G, forcing its arm deeper into the 30S subunit, which forces the peptidyl tRNA from the A site into the P site, carrying the mRNA and deacylated tRNA with it. Results in the ribosome moving along the
mRNA by length of one codon

Question 19

What does the probability of forming a protein with no erroes depend on?

Answer 19

The numebr of amino acids (n)
The frequency of insertion of a wrong amino acid (ε)
p=(1-ε)ⁿ

Question 20

How is elongation a circular process?

Answer 20

EF-Tu-GTP places the aminoacyl-tRNA on the ribosome and then is released as EF-Tu-GDP
EF-Ts is required to mediate the replacement of GDP by GTP
The reaction consumes GTP and releases GDP
The only aminoacyl-tRNA that cannot be recognised by EF-Tu-GTP is fMet-tRNAf, preventing use internally
Carries on cycling

Question 21

How is elongation terminated in prokaryotes?

Answer 21

1. Release factos RF1 (recognises stop codons UAA + UGA), RF2 (recognises stop codons UAA + UAG), RF3 (aids int he bidning)
2. RF binds to 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

Question 22

How is protein synthesis initiated in eukaryotes?

Answer 22

AUG is almost always used as the initiation codon
A special initiator tRNA, tRNAimet is used as the initiator (does not become formylated tRNA mmet is used to insert internal methionines)
The “first” AUG is usually used for initiation (~90%) but this is context dependent

Question 23

What is the Kozak sequence?

Answer 23

The sequence that gets the most effective recognition at the start codon
A(orG)xxAUGG (x can be any base)

Question 24

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

Answer 24

eIF4E binds 5’ cap
eIF-4A is an ATP-dependent RNA helicase that removes secondary structure
near the 5’ end - Needed for scanning movement of the 40S subunit along the mRNA
eIF-4G is a “scaffold” subunit and links together the initiation complex
Cleavage by protease results in inhibition of cap initiation

Question 25

What is the mechanism for the initiation of protein synthesis in eukaryotes?

Answer 25

Unlike prokaryotes, the Met-tRNAi is pre-bound to the 40S subunit, i.e. it’s binding is not codon-directed
The mRNA is scanned to find the first AUG
The ATP requirement is for the helicase activity of eIF-4A to remove hairpin structures in the mRNA

Question 26

How does elongation occur in eukaryotes?

Answer 26

PAB1(poly-(A) binding protein) interacts with eIF4G and eIF4E bound to “cap”
Ribosomes that have completed translation dissociate into subunits. These can readily find the nearby m⁷G cap and initiate another round of protein synthesis
Rapid recycling of subunits increases the efficiency of translation

Question 27

What are internal ribosome entry sites (IRES)?

Answer 27

The vast majority of eukaryotic mRNAs are translated through the ribosome scanning mechanism (90%)
An alternative mechanism is internal ribosome entry (10%)
The mRNAs lack a 5’ cap, and translation is initiated at internal ribosome entry sites (IRES)
IRESs have a complicated tertiary structure and bind 40S subunits in close proximity to an AUG codon

Question 28

How does mitosis effect proteins synthesis?

Answer 28

Decrease of 75% total protein synthesis ,caused by cell cycle-dependent
dephosphorylation of eIF-4E (Cap binding)
Cap-binding translation severely reduced
IRES-containing RNAs unaffected as IRES doesn’t require a cap

Question 29

How do mitosis picornaviruses proteins synthesis?

Answer 29

Shuts off ~90% of host protein synthesis by producing protease that clips part of eIF4G so can no longer associate with cap, resulting in only IRES translation
Gives virus maximum competition with the host
Viruses use IRES, so cell cycle independent

Question 30

What is the structure of the eukaryote release factor?

Answer 30

Mimics the structure of the amino acid 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 center of the ribosome. This water hydrolyses the ester bond of peptidyl tRNA releasing the polypeptide

Question 31

How is elongation terminated in eukaryotes?

Answer 31

The water molecule bound to the release factor hydrolyses the ester bond in the peptide tRNA, releasing the completed polypeptide
During normal chain elongation, water is excluded from the peptidyl transferase centre of the ribosome

Question 32

What are the translational control mechanisms?

Answer 32

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 mRN

Question 33

How is initiation controlled by eIF2a phosphorylation?

Answer 33

eIF2 is needed to bind to the methionine tRNA
eIF2 is phosphorylated in response to kinase cascades from external signals via signal transduction pathways
This phosphorylation blocks the exchange factor needed to recharge eIF2 with GTP, by blocking this with phosphorylation, you stop translation initiation

Question 34

How is initiation controlled by eIF4 phosphorylation?

Answer 34

eIF4E is a binding protein that if phosphorylated, cannot have 4E-binding proteins bind to it, allowing the eIF4E to function normally
If eIF4E is dephosphorylated, 4E-BPs bind to eIF4E, IF4E is tethered in such a way that it cannot start initiation (again controlled by what the cell is experiencing)

Question 35

What is the demand for ribosomal protein (r-protein) dependent on and closely coupled with?

Answer 35

The demand for r-proteins is growth rate dependent (high demand during rapid growth, low demand during slow growth). It is also closely coupled to rRNA synthesis

Question 36

How is r-protein synthesis regulated and how is this shown?

Answer 36

R-protein synthesis is regulated at the level of translation. Which is shown by transforming E. coli with a high copy plasmid encoding r-protein. mRNA level increases greatly, but not the amount of r-protein

Question 37

How are ribosomal protein genes organised?

Answer 37

Into several operons, each containing up to 11 genes for r-proteins. Some of these operons also encode non-ribosomal proteins whose synthesis is growth rate dependent

Question 38

When rRNA is in short supply (e.g when cells are nutrient limited), what happens to the levels of free r-proteins and how does this change occur?

Answer 38

Increases
One r-protein from each operon then binds to the polycistronic mRNA near to the ribosome binding site of one of the first genes of the operon. This prevents translation of this and the
other downstream ORFs of the operon
This is called autogenous (self-limiting) control

Question 39

How is translation of mammalian ferritin and transferrin mRNAs regulated by iron-response element binding proteins (cytosolic aconitase) (iron homeostasis)?

Answer 39

Ferritin is a cytosolic protein that binds iron ions and prevents accumulation of toxic levels of Fe²⁺/Fe³⁺
However when Fe is limiting, ferritin poses a problem – it competes for Fe with iron-requiring enzymes
Mammalian cells therefore modulate the synthesis of ferritin – expressed under excess Fe,
repressed under Fe scarcity
The transferrin receptor (a cell surface protein responsible for Fe uptake into cells) shows
reciprocal regulation of synthesis to that of ferritin

Question 40

What does cytosolic aconitase do?

Answer 40

Binds to IRE under iron starvation: ferritin translation blocked
Transferrin receptor mRNA stabilised by bound actinose: mRNA translated

Question 41

What does cytosolic aconitase do when there is excess iron ions?

Answer 41

Actinose undergoes conformation change on iron binding: IRE released ferritin mRNA is translated, transferrin receptor mRNA is degraded

Question 42

How does eukaryotic mRNA decay?

Answer 42

Nearly all mRNAs subjected to poly-(A) tail shortening
When tail < 30 A’s residues in length, Poly (A) binding protein is lost and 3’ end no longer
associates with cap
This leads to decapping followed by degradation

Question 43

How does cleavage at the endonuclease cleavage site in the 3’ UTR affect eukaryote mRNA decay?

Answer 43

Results in decapping followed by fast degradation