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Molecular Biology Fourth Edition

Molecular Biology Fourth Edition. Chapter 18 The Mechanism of Translation II: Elongation and Termination. Chapter 17 The Mechanism of Translation I: Initiation. Chapter 19 Ribosomes and Transfer RNA. Robert F. Weaver. 17.1 Initiation of Translation in Bacteria.

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Molecular Biology Fourth Edition

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  1. Molecular BiologyFourth Edition Chapter 18 The Mechanism of Translation II: Elongation and Termination Chapter 17 The Mechanism of Translation I: Initiation Chapter 19 Ribosomes and Transfer RNA Robert F. Weaver

  2. 17.1 Initiation of Translation in Bacteria • Two important events must occur before translation initiation can take place • Generate a supply of aminoacyl-tRNAs • Amino acids must be covalently bound to tRNAs • Process of bonding tRNA to amino acid is called tRNA charging • Dissociationof ribosomes into their two subunits • The cell assembles the initiation complex on the small ribosomal subunit • The two subunits must separate to make assembly possible

  3. tRNA Charging • All tRNAs have same 3 bases at 3’-end (CCA) • Terminal adenosine is the target for charging with amino acid • Amino acid attached by ester bond between • Its carboxyl group • 2’- or 3’-hydroxyl group of terminal adenosine of tRNA Amino acid

  4. Two-Step Charging • Aminoacyl-tRNA synthetases join amino acids to their cognate tRNAs • This is done in a two-step reaction: • Begins with activation of the amino acid with AMP derived from ATP • In the second step, the energy from the aminoacyl-AMP is used to transfer the amino acid to the tRNA

  5. Aminoacyl-tRNA Synthetase Activity AMP/amino acid coupling AMP/tRNA displacement

  6. Dissociation of Ribosomes • E. coli ribosomes dissociate into subunits at the end of each round of translation • IF1 actively promotes this dissociation • IF3 binds to free 30S subunit and prevents reassociation with 50S subunit to form a whole ribosome

  7. Ribosomal Subunit Exchange Grow in heavy isotope of nitrogen, carbon, and hydrogen. Then 3H labeled

  8. Formation of the 30S Initiation Complex When ribosomes have been dissociated into 50S and 30S subunits, cell builds a complex on the 30S subunit: • mRNA • Aminoacyl-tRNA • Initiation factors • IF3 binds by itself to 30S subunit • IF1 and IF2 stabilize this binding • IF2 can bind alone, but is stabilized with help of IF1 and IF3 • IF1 does not bind alone

  9. First Codon and the First Aminoacyl-tRNA • Prokaryotic initiation codon is: • Usually AUG • Can be GUG • Rarely UUG • Initiating aminoacyl-tRNA is N-formyl-methionyl-tRNA • N-formyl-methionine (fMet) is the first amino acid incorporated into a polypeptide • This amino acid is frequently removed from the protein during maturation

  10. N-Formyl-Methionine Lipman et al., Marcker and Sanger

  11. Formyl-Met-tRNA and Met-tRNA • Which codons they respond to? • Which position within the protein they placed methionine?

  12. Which codons they respond to? • Make a labeled aminoacyl-tRNA, mix it with ribosomes and a variety of trinucleotides, such as AUG. • Met-tRNA bind AUG, formyl-Met-tRNA binds AUG, GUG, and UUG. AUG >90% , GUG about 8% UUG 1%

  13. Which position within the protein they placed methionine? • mRNA Sequence AUG AUG AUG……. • In vitro translation system • In the presence of tRNAmet, met is incorporated into interior of the product • In the presence of formy-tRNAmet, met is incorporated into the first codon of the product

  14. Weigert and Garen:Formyl-Met of the the polypeptide is always removed in bacteria and phage proteins

  15. Binding mRNA to the 30S Ribosomal Subunit • The 30S initiation complex is formed from a free 30S ribosomal subunit plus mRNA and fMet-tRNA • Binding between the 30S prokaryotic ribosomal subunit and the initiation site of a message depends on base pairing between • Short RNA sequence • Shine-Dalgarno sequence • Upstream of initiation codon • Complementary sequence • 3’-end of 16S RNA

  16. Binding mRNA and fMet-tRNA to the 30S ribosomal subunit How does the cell detect the difference between the initiation codon and an ordinary codon with the same sequences? Consensus sequences?

  17. Positive strand phages • R17 • f2 • MS2 • Positive strand phages • Encode three genes: A (maturation) protein, coat protein and replicase

  18. Gene structure of MS2 phage RNA

  19. The secondary structures have inhibitory effect

  20. Lodish et al: • Translating f2 coat mRNA by ribosomes from different bacteria • B. stearothermophilus could only translate A protein but not coat protein • (This is not due to initiation factors but ribosomes)

  21. Nomura et al., • The important element is in the 30S ribosome • If the 30S ribosome from the E. coli, the coat protein can be translated • If the 30S ribosome from the B. stearothermophilus, the coat protein can not be translated • The active elements are S12 and 16S rRNA

  22. Shine and Dalgarno • Upstream of initiation codon: 5’- AGGAGGU • 3’ end of the 16S rRNA:3’HO-AUUCCUCCAC • B. stearothermophilus has poor match

  23. Bacillus 16S rRNA • 4 Watson Crick base pairing with the A protein and replicase ribosome binding sites • 2 with the coat protein gene • E. coli • At least three base pairs with all three genes Could the base pairing between 16S rRNA and the region upstream of the translation initiation site be vital to ribosome binding?

  24. Shine-Dalgarno (SD) sequence See Table 17.1 AGGAGGU From ribosomes from C. crescentus and P. aeruginosa No ribosome binding would occur for less than 3 bp

  25. Steitz and Jakes • Ribosome from E. coli bound to initiation site and treated with Colicin E3 (RNase) • Fingerprinting • Initiation site including S-D sequence • An oligonucleotide from 3’end of the 16S rRNA

  26. The best evidence • Hui and De Boer in 1987 • Clone the human growth hormone gene into E. coli

  27. Initiation Factors and 30S Subunit • Binding of the Shine-Dalgarno sequence with the complementary sequence of the 16S rRNA is mediated by IF3 • Assisted by IF1 and IF2 • All 3 initiation factors have bound to the 30S subunit at this time

  28. Binding of fMet-tRNA to the 30S Initiation Complex • IF2 is the major factor promoting binding of fMet-tRNA to the 30S initiation complex • Two other initiation factors also play an important supporting role • GTP is also required for IF2 binding at physiological IF2 concentrations • The GTP is not hydrolyzed in the process

  29. 30S Initiation Complex The complete 30S initiation complex contains one each: • 30S ribosomal subunit • mRNA • fMet-tRNA • GTP • Factors IF1, IF2, IF3

  30. Formation of the 70S Initiation Complex • GTP is hydrolyzed after the 50S subunit joins the 30S complex to form the 70S initiation complex • This GTP hydrolysis is carried out by IF2 in conjunction with the 50S ribosomal subunit • Hydrolysis purpose is to release IF2 and GTP from the complex so polypeptide chain elongation can begin

  31. What is the function of GTP hydrolysis GTP hydrolysis is to remove IF-2 from the ribosomes Exp: 30S initiation complex+labeled IF-2 and fMet-tRNA and either GDPCP or GTP, add 50S and ultracentrifugation

  32. 17.18 Effect of GTP hydrolysis on release of IF-2 from the ribosome. After adding 50S, IF-2 is released from the 70S ribosome but fmet-tRNA is still associated

  33. In Fig 17.18, more fMet-tRNA is associated with the 70S ribosomes • Catalytic function of IF2 • Hydrolysis of GTP is necessary to release IF2 from the 70S initiation complex so it can bind another molecule of fmet-tRNA, otherwise, IF2 only acts stoichiometrically

  34. Bacterial Translation Initiation • IF1 influences dissociation of 70S ribosome to 50S and 30S • Binding IF3 to 30S, prevents subunit reassociation • IF1, IF2, GTP bind alongside IF3 • Binding mRNA to fMet-tRNA forming 30S initiation complex • Can bind in either order • IF2 sponsors fMet-tRNA • IF3 sponsors mRNA • Binding of 50S with loss of IF1 and IF3 • IF2 dissociation and GTP hydrolysis

  35. Eukaryotic Begins with methionine Initiating tRNA not same as tRNA for interior No Shine-Dalgarno mRNA have caps at 5’end Bacterial N-formyl-methionine Shine-Dalgarno sequence to show ribosomes where to start 17.2 Initiation in Eukaryotes

  36. Scanning Model of Initiation • Eukaryotic 40S ribosomal subunits locate start codon by binding to 5’-cap and scanning downstream to find the 1st AUG in a favorable context • Kozak’s Rules are a set of requirements • Best context uses A of ACCAUGG as +1: • Purine (A or G) in -3 position • G in +4 position • 5-10% cases ribosomal subunits bypass 1st AUG scanning for more favorable one

  37. The scanning model of initiation Kozak systematically mutated nucleotides around the initiation ↓ codon in a cloned rat preproinsulin gene ↓ Introduce into COS cells ↓ Label newly synthesized protein with 35S-Met ↓ Immunoprecipitate ↓ Electrophoresis ↓ Detect by autoradipgraph

  38. The scanning model of initiation The better the translation initiation, the more proinsulin was made

  39. The best initiation occur with a G or an A in position –3 and a G in position +4 (where the A in ATG is position +1) A/G C CA T G G-3 -2 -1 +1 +2 +3 +4

  40. Figure 17.21 Effects of single base changes in positions –3 and +4 surrounding the initiating ATG.

  41. If this really is the optimum sequence for translation initiation, introducing it out of frame and upstream of the normal initiation codon should provide a barrier to scanning ribosomes and force them to initiate out of frame

  42. Out-of-frame ATG The closer it resembled the optimal sequence, the more it interfered with initiation at the downstream ATG

  43. Translation With a Short ORF • Ribosomes can use a short upstream open reading frame: • Initiate at an upstream AUG • Translate a short Open Reading Frame • Continue scanning • Reinitiate at a downstream AUG

  44. Scanning Model for Translation Initiation

  45. Effects of mRNA Secondary Structure • Secondary structure near the 5’-end of an mRNA can have either positive or negative effects • Hairpin just past an AUG can force a pause by ribosomal subunit and stimulate translation • Very stable stem loop between cap and initiation site can block scanning and inhibit translation

  46. Eukaryotic Initiation Factors • Bacterial translation initiation requires initiation factors as does eukaryotic initiation of translation • Eukaryotic system is more complex than bacterial • Scanning process • Factors to recognize the 5’-end cap

  47. Translation Initiation in Eukaryotes Eukaryotic initiation factors and general functions: • eIF2 binds Met-tRNA to ribosomes • eIF2B activates eIF2 replacing its GDP with GTP • eIF1 and eIF1A aid in scanning to initiation codon • eIF3 binds to 40S ribosomal subunit, inhibits reassociation with 60S subunit • eIF4 is a cap-binding protein allowing 40S subunit to bind 5’-end of mRNA • eIF5 encourages association between 60S ribosome subunit and 48S complex • eIF6 binds to 60S subunit, blocks reassociation with 40S subunit Fig. 17.22

  48. Function of eIF4 • eIF4 is a cap-binding protein • This protein is composed of 3 parts: • eIF4E, 24-kD, has actual cap binding activity • eIF4A, a 50-kD polypeptide • eIF4G is a 220-kD polypeptide • The complex of the three polypeptides together is called eIF4F

  49. The cap-binding proteins GDP-binding protein Cap binding protein

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