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Two Hybrid

Two Hybrid. Chapter 24 Translation. 24.1 Introduction. Figure 24.02: Ribosomes are large ribonucleoprotein particles that contain more RNA than protein and dissociate into large and small subunits. Figure 24.01: Size comparisons show that the ribosome is large enough to bind tRNAs and mRNA.

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Two Hybrid

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  1. Two Hybrid

  2. Chapter 24Translation

  3. 24.1 Introduction Figure 24.02: Ribosomes are large ribonucleoprotein particles that contain more RNA than protein and dissociate into large and small subunits. Figure 24.01: Size comparisons show that the ribosome is large enough to bind tRNAs and mRNA.

  4. 24.2 Translation Occurs by Initiation, Elongation, and Termination • The ribosome has three tRNA-binding sites. • An aminoacyl-tRNA enters the A site. • Peptidyl-tRNA is bound in the P site. • Deacylated tRNA exits via the E site. Figure 24.03: The ribosome has two sites for binding charged tRNA.

  5. 24.2 Translation Occurs by Initiation, Elongation, and Termination • An amino acid is added to the polypeptide chain by transferring the polypeptide from peptidyl-tRNA in the P site to aminoacyl-tRNA in the A site (translocation). Figure 24.05: Aminoacyl-tRNA enters the A site, receives the polypeptide chain from peptidyl-tRNA, and is transferred into the P site.

  6. 24.2 Translation Occurs by Initiation, Elongation, and Termination • initiation – The stages of translation up to synthesis of the first peptide bond of the polypeptide. • elongation – The stage of translation in which the polypeptide chain is extended by the addition of individual subunits. • termination – A separate reaction that ends translation by stopping the addition of subunits and (typically) causing disassembly of the synthetic apparatus.

  7. 24.2 Translation Occurs by Initiation, Elongation, and Termination Figure 24.07: Translation has three stages.

  8. 24.3 Special Mechanisms Control the Accuracy of Translation • The accuracy of translation is controlled by specific mechanisms at each stage. Figure 24.08: Errors occur at rates from 10–6 to 5 10–4 at different stages of translation.

  9. Transfer RNAs (tRNAs) • tRNAs are adapters between amino acids and the codons in mRNA molecules. • The anticodon of the tRNA base pairs with the codon of mRNA. • The amino acid is covalently attached to the 3’ end of the tRNA. • tRNAs often contain modified nucleosides.

  10. What is Inosine?

  11. Inosine

  12. The Wobble Hypothesis:Base-Pairing Involving the Third Base of the Codon is Less Stringent.

  13. Base-Pairing with Inosine at the Wobble Position

  14. Suppressor Mutations • Some mutations in tRNA genes alter the anticodons and therefore the codons recognized by the mutant tRNAs. • These mutations were initially detected as suppressor mutations that suppressed the effects of other mutations. • Example: tRNA mutations that suppress amber mutations (UAG chain-termination mutations) in the coding sequence of genes.

  15. Making a (UAG) Mutation

  16. Translation of an amber (UAG) Mutation in the Absence of a Suppressor tRNA

  17. Translation of an amber Mutation in the Presence of a Suppressor tRNA Note it is amber su3…why?????????

  18. Translation of an amber Mutation in the Presence of a Suppressor tRNA If there was a single tRNATyr gene, then could one have a amber supressor of it?

  19. 24.4 Initiation in Bacteria Needs 30S Subunits and Accessory Factors • ribosome-binding site – A sequence on bacterial mRNA that includes an initiation codon that is bound by a 30S subunit in the initiation phase of polypeptide translation. Figure 24.09: Initiation requires free ribosome subunits.

  20. 24.4 Initiation in Bacteria Needs 30S Subunits and Accessory Factors • Shine–Dalgarno sequence – The polypurine sequence AGGAGG centered about 10 bp before the AUG initiation codon on bacterial mRNA. • It is complementary to the sequence at the 3′ end of 16S rRNA.

  21. 24.4 Initiation in Bacteria Needs 30S Subunits and Accessory Factors • Initiation of translation requires separate 30S and 50S ribosome subunits. • Initiation factors (IF-1, IF-2, and IF-3), which bind to 30S subunits, are also required. Figure 24.10: Initiation factors stabilize free 30S subunits and bind initiator tRNA to the 30S-mRNA complex.

  22. 24.4 Initiation in Bacteria Needs 30S Subunits and Accessory Factors • A 30S subunit carrying initiation factors binds to an initiation site on mRNA to form an initiation complex. • IF-3 must be released to allow 50S subunits to join the 30S-mRNA complex. Figure 24.11: Initiation requires 30S subunits that carry IF-3.

  23. 24.5 Initiation Involves Base Pairing Between mRNA and rRNA • An initiation site on bacterial mRNA consists of the AUG initiation codon preceded by the Shine–Dalgarno polypurine hexamer ~10 bases upstream. • The rRNA of the 30S bacterial ribosomal subunit has a complementary sequence that base pairs with the Shine–Dalgarno sequence during initiation.

  24. 24.5 Initiation Involves Base Pairing Between mRNA and rRNA Figure 24.12: Ribosome-binding sites on mRNA can be recovered from initiation complexes.

  25. 24.6 A Special Initiator tRNA Starts the Polypeptide Chain • Translation starts with a methionine amino acid usually encoded by AUG. • Different methionine tRNAs are involved in initiation and elongation.

  26. 24.6 A Special Initiator tRNA Starts the Polypeptide Chain • N-formyl-methionyl-tRNA (tRNAfMet) – The aminoacyl-tRNA that initiates bacterial polypeptide translation. • The amino group of the methionine is formylated.

  27. 24.6 A Special Initiator tRNA Starts the Polypeptide Chain Figure 24.14: The initiator N-formyl-methionyl-tRNA is generated by formylation of methionyl-tRNA, using formyl-tetrahydrofolate as cofactor. Figure 24.15: fMet-tRNAf has unique features that distinguish it as the initiator tRNA.

  28. 24.6 A Special Initiator tRNA Starts the Polypeptide Chain • tRNAmMet– The bacterial tRNA that inserts methionine at internal AUG codons. • The initiator tRNA has unique structural features that distinguish it from all other tRNAs. • The NH2 group of the methionine bound to bacterial initiator tRNA is formylated.

  29. 24.7 Use of fMet-tRNAf Is Controlled by IF-2 and the Ribosome • context – The fact that neighboring sequences may change the efficiency with which a codon is recognized by its aminoacyl-tRNA or is used to terminate polypeptide translation. Figure 24.16: Only fMet-tRNAf can be used for initiation by 30S subunits; other aminoacyl-tRNAs must be used for elongation by 70S ribosomes.

  30. 24.7 Use of fMet-tRNAf Is Controlled by IF-2 and the Ribosome • IF-2 binds the initiator fMet-tRNAf and allows it to enter the partial P site on the 30S subunit. Figure 24.17: IF-2 is needed to bind fMet-tRNAf to the 30S-mRNA complex. After 50S binding, all IF factors are released and GTP is cleaved.

  31. 24.8 Small Subunits Scan for Initiation Sites on Eukaryotic mRNA • Eukaryotic 40S ribosomal subunits bind to the 5′ end of mRNA and scan the mRNA until they reach an initiation site. • A eukaryotic initiation site consists of a 10-nucleotide sequence that includes an AUG codon. • 60S ribosomal subunits join the complex at the initiation site.

  32. 24.8 Small Subunits Scan for Initiation Sites on Eukaryotic mRNA Figure 24.18: Eukaryotic ribosomes migrate from the 5 end of mRNA to the initiation site, which includes an AUG initiation codon.

  33. 24.8 Small Subunits Scan for Initiation Sites on Eukaryotic mRNA • internal ribosome entry site (IRES) – A eukaryotic messenger RNA sequence that allows a ribosome to initiate polypeptide translation without migrating from the 5′ end.

  34. 24.9 Eukaryotes Use a Complex of Many Initiation Factors • Initiation factors are required for all stages of initiation, including binding the initiator tRNA, 40S subunit attachment to mRNA, movement along the mRNA, and joining of the 60S subunit. • Eukaryotic initiator tRNA is a Met-tRNA that is different from the Met-tRNA used in elongation, but the methionine is not formylated. Figure 24.19: Some initiation factors bind to the 40S ribosome subunit to form the 43S preinitiation complex; others bind to mRNA.

  35. 24.9 Eukaryotes Use a Complex of Many Initiation Factors • eIF2 binds the initiator Met-tRNAi and GTP, forming a ternary complex that binds to the 40S subunit before it associates with mRNA. • A cap-binding complex binds to the 5′ end of mRNA prior to association of the mRNA with the 40S subunit. Figure 24.21: Initiation factors bind the initiator Met-tRNA to the 40S subunit to form a 43S complex.

  36. 24.10 Elongation Factor Tu Loads Aminoacyl-tRNA into the A Site • EF-Tu (an elongation factor) is a monomeric G protein whose active form (bound to GTP) binds to aminoacyl-tRNA. • The EF-Tu-GTP-aminoacyl-tRNA complex binds to the ribosome’s A site. Figure 24.25: EF-Tu-GTP places aminoacyl-tRNA on the ribosome and then is released as EF-Tu-GDP.

  37. 24.10 Elongation Factor Tu Loads Aminoacyl-tRNA into the A Site • GMP-PCP – An analog of GTP that cannot be hydrolyzed. • It is used to test which stage in a reaction requires hydrolysis of GTP. • kirromycin – An antibiotic that inhibits protein synthesis by acting on EF-Tu.

  38. 24.11 The Polypeptide Chain Is Transferred to Aminoacyl-tRNA • The 50S subunit has peptidyl transferase activity as provided by an rRNA ribozyme. • The nascent polypeptide chain is transferred from peptidyl-tRNA in the P site to aminoacyl-tRNA in the A site. • Peptide bond synthesis generates deacylated tRNA in the P site and peptidyl-tRNA in the A site.

  39. 24.11 The Polypeptide Chain Is Transferred to Aminoacyl-tRNA Figure 24.26: Peptide bond formation.

  40. 24.11 The Polypeptide Chain Is Transferred to Aminoacyl-tRNA • puromycin – An antibiotic that terminates protein synthesis by mimicking a tRNA and becoming linked to the nascent protein chain. Figure 24.27: Puromycin mimics aminoacyl-tRNA because it resembles an aromatic amino acid linked to a sugar-base moiety.

  41. 24.12 Translocation Moves the Ribosome • Ribosomal translocation moves the mRNA through the ribosome by three bases. • Translocation moves deacylated tRNA into the E site and peptidyl-tRNA into the P site and empties the A site. Figure 24.28: A bacterial ribosome has three tRNA-binding sites.

  42. 24.12 Translocation Moves the Ribosome • The hybrid state model proposes that translocation occurs in two stages, in which the 50S moves relative to the 30S and then the 30S moves along mRNA to restore the original conformation. Figure 24.29: Models for translocation involve two stages.

  43. 24.13 Elongation Factors Bind Alternately to the Ribosome • Translocation requires EF-G, whose structure resembles the aminoacyl-tRNA-EF-Tu-GTP complex. • Binding of EF-Tu and EF-G to the ribosome is mutually exclusive. • Translocation requires GTP hydrolysis, which triggers a change in EF-G, which in turn triggers a change in ribosome structure.

  44. 24.13 Elongation Factors Bind Alternately to the Ribosome Figure 24.30: Binding of factors EF-Tu and EF-G alternates as ribosomes accept new aminoacyl-tRNA, form peptide bonds, and translocate.

  45. 24.14 Three Codons Terminate Translation • The stop codons UAA (ochre), UAG (amber), and UGA (opal) terminate translation. • In bacteria, they are used most often with relative frequencies UAA>UGA>UAG.

  46. 24.14 Three Codons Terminate Translation • premature termination – The termination of protein or of RNA synthesis before the chain has been completed. • In translation it can be caused by mutations that create stop codons within the coding region. • In RNA synthesis it is caused by various events that act on RNA polymerase.

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