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LF206: Molecular Cell Biology > Lecture 9:  The mechanics of translation > Flashcards

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
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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
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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
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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
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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
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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
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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
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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
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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
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10
Q

What does the binding of incoming aminoacyl-tRNA require?

A
  • Soluble supernatant factor
  • Elongation factor T (EF-T)
  • GTP
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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
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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.)
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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
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14
Q

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

A
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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
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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
    • RESULT: fully activated ribosome
      • (Does not become formylated)
      • tRNAmmet is used to insert internal methionines.
    • The “first” AUG is usually use for initiation (~90%).
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.
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
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
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
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.

LF206: Molecular Cell Biology (16 decks)

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