Lecture 9: The mechanics of translation

Question 1

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

Answer 1

• Eukaryotic mRNA: monocistronic
  ○ Poly A tail
  
  • Prokaryotic mRNA: polycistronic (more than one gene often on 1 string of mRNA)
    ○ Specific ribosome binding site (RBS)
    ○ No Poly A tail
    ○ Shine-Dalgarno (SD) sequence

Question 2

What is the start sequence for prokaryotic translation?

Answer 2

• AUG (or GUG) preceded by several bases that pair w 16S rRNA
  
  • Shine-Dalgarno sequence
    ○ Purine-rich
    ○ Complementary to initiator sites of mRNA

Question 3

What are the two types of tRNAmet found in E.coli?

Answer 3

• tRNAf
  ○ Met residues attached to this are formylated
  ○ Initiate polypeptide chains only
  ○ Recognizes AUG n GUG
  § GUG internally codes for valine
  
  • tRNAm
    ○ Met residues are only attached, not formylated
    ○ Recognizes the codon AUG only
    ○ Used as a source of internal Met residues

Question 4

Describe an experiment illustrating the evidence of protein ‘factors’ in initiation

Answer 4

• Protein synthesis initiation in bacteria requires free 30S subunits
  
  • 30S subunit are washed in high salt (e.g. 0.5M KCl) -> subunits lose their ability to initiate protein synthesis
  • Supernatant from high salt dialyzed to remove salt
  • Salt added back to the salt-washed 30S subunit -> activity restored
  • Chromatography of high salt supernatant reveal that 3 protein factors are necessary for initiation
    IF1, IF2, IF3

Question 5

Describe the 30S initiation complex for translation initiation in prokaryotes

Answer 5

• IF-3
  ○ 22kDA
  ○ Binds to the 30S subunit to prevent premature association w the 50S subunit -> inhibits full active ribosome complex
  
  • IF1
    ○ 9kDa
    ○ Sits at A pocket (where the incoming tRNAs will first choose to reside)
    ○ Forces fmet-tRNA to go to the P pocket, where it needs to be for the initiation
  • GTP hydrolysis n dissociation of initiation factors that allow the last subunit to come n sit on the mRNA w a …. Attached
  • IF-2
    ○ 120kDa
    ○ Reacts w fmet-tRNA n GTP to form ternary complex IF-2-GTP-fmet-tRNA
    § Guides ternary complex to partial P site in the 30S subunit-mRNA complex
    § Triggers GTP hydrolysis when the 50S subunit joins the complex
    Does not recognize met-tRNA or any aa tRNA used for elongation

Question 6

Describe what makes up the 70S initiation complex in prokaryotes n what does it do?

Answer 6

• 50S subunit + 30S subunit = 70S initiation complex
  
  • Initiation factors released
  • GTP hydrolyzed

Question 7

What are the 3 steps of elongation in protein synthesis?

Answer 7

• Codon directed binding of the incoming aminoacyl-tRNA
  
  • Peptide bond formation
  • Translocation (movement) of the ribosome along w mRNA in 5’->3’ direction by the length of 1 codon

Question 8

Describe peptide bond formation in the context of elongation in protein synthesis

Answer 8

• Hydrophilic movement
  ○ H+ is lost -> tetrahedral intermediate formed -> -OH on P site [H+ comes back in, which hydrolyzes tRNA at P site, releasing tRNA]
  
  • Aminoacyl portion of fMet-tRNA is transferred to the aa group of the aa residue in the A site -> peptide bond
  • Bc the tRNAse hv got high energy bond holding AA to tRNA, the actual energy required to carry out this activity in the ribosome is 0
    ○ All the energy is stored in the bonds of the dissociating tRNAs

Question 9

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

Answer 9

• Elongation factors attached to tRNA
  
  • Move to vacant A site
  • Proofreading step
    ○ Make sure right AA attached to right tRNA that is recognizing the right codon
  • GTP hydrolysis: allows for reorganization of the tRNA to allow it to come into close contact with the tRNA in the P site
    ○ Allows peptide transferase to occur
  • Chain transferring to A site
  • EF-G facilitates translocation of ribosome along mRNA
    ○ Slots into a partial A site
  • Switch protein
    ○ Switches when GTP is hydrolyzed
    ○ Forces itself into the rest of the A site -> pulls ribosome along by 1 codon
  • All these things hv to hv similar shapes that allow them to fit into the same pockets n make similar interactions
  • GTP gets hydrolyzed to GDP
  • Fully empty A site, peptide now in P site
  • Previous tRNA that lost its cargo, sat on exit site -> cycle repeats
  • Lecture 8: diphtheria toxin acts on the elongation step

Question 10

What does the binding of incoming aminoacyl-tRNA require?

Answer 10

• Soluble supernatant factor
• Elongation factor T (EF-T)
• GTP

Question 11

What is EF-T composed of?

Answer 11

• EF-T composed of 2 polypeptides
  ○ EF-Tu (45kDA, heat unstable)
  * Abundant (~20 mols per ribosome)
  ○ Most aa-tRNAs in the cell are complexed w EF-Tu
  * Ester bond is very high energy and susceptible to hydrolysis so needs to be protected until it fits into the peptide pocket
  * Does not react w met-tRNAmet -> fmet is never inserted in the protein
  ○ EF-Ts (30kDa, heat stable)
  * Mediates the replacement of GDP by GTP
  
  • When bound to EF-Tu, labile ester bond b/w the tRNA n aminoacyl residue is protected from hydrolysis

Question 12

How does EF-Tu proofread in prokaryotic translation?

Answer 12

• Proofreading is important to maintain fidelity b/w intended sequences in mRNA n actual sequences
  
  • When GTP is hydrolyzed
    ○ EF-Tu undergoes a conformational change (few milliseconds process)
    ○ Gives system time to see whether there’s a good interaction b/w codon n anticodon
  • Intervals between the hydrolysis and release of GDP -> allows time for weakly bound, non-cognate aa-tRNA (i.e. incorrect codon-anticodon match) to dissociate from the ribosome
  • If not sitting properly -> rejected (no insertion of the wrong a.a.)

Question 13

“How do the structural differences between EF-Tu and EF-G affect their roles in ribosome translocation during translation?”

Answer 13

• EF Tu attached to tRNA is a mixture of RNA n protein
  
  • EF G is all protein
  • Similar shape, distribution of charges n polar regions
    ○ This allows EF-G to fit into pockets n affects translocation process of ribosome

Question 14

What are the main differences between prokaryotic and eukaryotic initiation factors?

Question 15

What is themechanism of translocation in elongation during prokaryotic translation?

Answer 15

• EF-G +GTP complex 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.
    RESULT:-ribosome moves along the mRNA by length of one codon

Question 16

How do you calculate the probability of forming a protein with no errors?

Answer 16

p = (1-e)n

n = number of a.a.
e = the frequency of insertion of the wrong a.a.

Question 17

Describe how prokaryotic termination occurs

Answer 17

• Release factors recognize stop codons
  ○ RF1: UAA + UGA
  ○ RF2: UAA + UAG
  ○ RF3 (switch protein): GTP aids binding
  
  • RF binds to vacant A site
  • Peptidyl transfer of the peptidyl group to water rather than aminoacyl tRNA
  • Polypeptide is released
  • Hydrolysis of RF3
    ○ GTP -> GDP
    GDP n RF3 released

Question 18

What is involved in initiation of protein synthesis in eukaryotes?

Answer 18

• AUG is almost always used as the initiation codon.
  
  • A special initiator tRNA, tRNAimet is used as the initiator.
    
    • tRNAimet prebound to 40S subunit
      § eIF1 guides tRNAimet to the 40S subunit
      § eIF2 binds
    • mRNA is scanned to the first AUG using eIF-4B
    • Switch proteins rearrnage -> allows larger ribosomal subunit to bind
  • RESULT: fully activated ribosome
    
    • (Does not become formylated)
    • tRNAmmet is used to insert internal methionines.
  • The “first” AUG is usually use for initiation (~90%).

Question 19

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

Answer 19

• eIF4E binds to the 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
    ○ Links together the initiation complex .
    ○ Cleavage by protease -> inhibition of cap initiation.

Question 20

How is regulation of protein synthesis during the cell cycle?

Answer 20

• G2/M transition
  ○ 75% total protein synthesis
  ○ Caused by cell cycle-dependent dephosphorylation of eIF-4E (cap binding)
  ○ Decreases affinity of ribosomes for the cap
  
  • IRES-containing RNAs are unaffected
    ○ Relative IRES translation rates increase in M phase
  • Apoptosis
    ○ eIF-4G is cleaved (capsase 3)
    § All translation decreases

Question 21

What is the structure of a eukaryotic release factor?

Answer 21

• Mimics the structure of the AA acceptor stem of tRNA (CCA terminus)
  
  • Gly-Gly-Gln at the tip of the acceptor stem binds a water molecule
  • This is carried into the peptidyl transferase center of the ribosome
  • Water hydrolyzes ester bond of peptidyl tRNA releasing the polypeptide

Question 22

How are polypeptide chains released during eukaryotic translation?

Answer 22

• Water molecule bound to the release factor hydrolyzes the ester bond in the peptidyl tRNA -> release complete polypeptide
  
  • During normal chain elongation, water is excluded from the peptidyl transferase center of the ribosome

Question 23

What are the translational control mechanisms?

Answer 23

• 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 24

How can eIF2a phosphorylation control initiation?

Question 25

Describe the autogenous control of ribosomal protein synthesis in E. coli

Answer 25

* Ribosomal (r) proteins ○ Growth dependent ○ Closely coupled with rRNA synthesis * When cells are nutrient limited ○ rRNA is in short supply ○ Levels of free r-proteins increase ○ One r-protein from each operon binds to polycistronic mRNA near the ribosome binding site Prevents translation of the gene downstream

Question 26

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

Answer 26

* Ferritin: cytosolic protein that binds iron ions n prevents accumulation * When Fe is limiting, ferritin poses a problem ○ Competes for Fe w ironing-requiring enzymes * Modulate ferritin synthesis [mammalian cells] ○ Expressed under excess Fe ○ Repressed under Fe scarcity * Transferrin receptor (cell surface protein responsible for Fe uptake into cells) shows reciprocal regulation of synthesis to that of ferritin * Aconitase (Iron response element (IRE) binding protein) binds Fe ○ Conformational change ○ IRE is released ○ Ferritin mRNA is translated ○ Transferrin receptor mRNA is degraded

Question 27

What is involved in eukaryotic mRNA decay?

Answer 27

* All mRNAs subjected to poly (A) tail shortening * When tail <30 A's residues in length, Poly (A) binding protein is lost -> 3' end no longer associates w cap * RESULT: decapping followed by degradation ○ Cleavage at the endonuclease cleavage site in the 3’ UTR -> decapping followed by degradation.