Lecture 9: The mechanics of translation Flashcards
1
Q
How do the initiation and termination regions differ in eukaryotic and prokaryotic mRNA?
A
- 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
- Prokaryotic mRNA: polycistronic (more than one gene often on 1 string of mRNA)
2
Q
What is the start sequence for prokaryotic translation?
A
- AUG (or GUG) preceded by several bases that pair w 16S rRNA
- Shine-Dalgarno sequence
○ Purine-rich
○ Complementary to initiator sites of mRNA
- Shine-Dalgarno sequence
3
Q
What are the two types of tRNAmet found in E.coli?
A
- 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
- tRNAm
4
Q
Describe an experiment illustrating the evidence of protein ‘factors’ in initiation
A
- 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
5
Q
Describe the 30S initiation complex for translation initiation in prokaryotes
A
- 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
- IF1
6
Q
Describe what makes up the 70S initiation complex in prokaryotes n what does it do?
A
- 50S subunit + 30S subunit = 70S initiation complex
- Initiation factors released
- GTP hydrolyzed
7
Q
What are the 3 steps of elongation in protein synthesis?
A
- 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
8
Q
Describe peptide bond formation in the context of elongation in protein synthesis
A
- 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
9
Q
What is the mechanism of translocation in elongation during prokaryotic translation?
A
- 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
10
Q
What does the binding of incoming aminoacyl-tRNA require?
A
- Soluble supernatant factor
- Elongation factor T (EF-T)
- GTP
11
Q
What is EF-T composed of?
A
- 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
12
Q
How does EF-Tu proofread in prokaryotic translation?
A
- 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.)
- When GTP is hydrolyzed
13
Q
“How do the structural differences between EF-Tu and EF-G affect their roles in ribosome translocation during translation?”
A
- 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
14
Q
What are the main differences between prokaryotic and eukaryotic initiation factors?
A
15
Q
What is themechanism of translocation in elongation during prokaryotic translation?
A
- 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
16
Q
How do you calculate the probability of forming a protein with no errors?
A
p = (1-e)n
n = number of a.a.
e = the frequency of insertion of the wrong a.a.
17
Q
Describe how prokaryotic termination occurs
A
- 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
18
Q
What is involved in initiation of protein synthesis in eukaryotes?
A
- 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
- tRNAimet prebound to 40S subunit
- RESULT: fully activated ribosome
- (Does not become formylated)
- tRNAmmet is used to insert internal methionines.
- The “first” AUG is usually use for initiation (~90%).
- A special initiator tRNA, tRNAimet is used as the initiator.
19
Q
What is in the cap binding complex (elF-4F) in eukaryotes?
A
- 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.
- eIF-4A is an ATP-dependent RNA helicase that removes secondary structure near the 5’ end.
20
Q
How is regulation of protein synthesis during the cell cycle?
A
- 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
- IRES-containing RNAs are unaffected
21
Q
What is the structure of a eukaryotic release factor?
A
- 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
22
Q
How are polypeptide chains released during eukaryotic translation?
A
- 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
23
Q
What are the translational control mechanisms?
A
- 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
24
Q
How can eIF2a phosphorylation control initiation?
A
25
Q
Describe the autogenous control of ribosomal protein synthesis in E. coli
A
- 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
- When cells are nutrient limited
26
Q
How is the translation of mammalian ferritin and transferrin mRNAs regulated by iron-response element binding proteins?
A
- 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
- When Fe is limiting, ferritin poses a problem
27
Q
What is involved in eukaryotic mRNA decay?
A
- 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.
