Chapter 18:The Mechanism of Translation : Elongation and Termination

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Chapter 18The Mechanism of Translation ?
Elongation and Termination

• ???

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Content

• 18.1 The direction of polypeptide synthesis
  and of mRNA translation
• 18.2 The genetic code
• 18.3 The elongation mechanism
• 18.4 Termination
• 18.5 Post termination

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If We want to learn about the nature of
elongation,We would ask

• In What direction is a polypeptide synthesized?
• Or Do protein chains grow in the
  amino-to-carboxyl direction ,or the reverse?
• Or Which amino acid is inserted first into a
  growing polypeptide

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Experiment strategy to determine the direction of
translation(Figure18.1)

• 1.Label the growing globin chains for various
  short lengths of time with Hleucine
• 2.Resultlabeling is strongest in
  carboxyl-terminal peptides

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Determining the direction of translation
(Figure18.2)

• 1.Plot the relative amount of H label
• With the N terminal peptide on the left , and the
  C-terminal peptide on the right
• 2.Result The curves showed the most label in
  the C-terminal peptides , especially after short
  labeling times

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• From the experiments above , We can conclude that
  the translation direction is amino---carboxyl
  direction

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• 1? In What direction does the
• ribosome read the mRNA?
• or Is the mRNA read in the 5-3-direction or
  the reverse ?

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• In the 1960s , Ochoa translated the mRNA
• 5-AUGUUU-3,They obtained fMet-phe ,
• Where the fMet was at the amino terminus
• AUG----fMet UUU----phe
• Therefore the mRNA must have been read from the
  5-end

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Summary

• Messenger RNAs are read in the 5-3direction
  ,the same direction in which they are synthesized
  .
• Proteins are made in the amino-carboxyl direction
  , which means that the amino terminal amino acid
  is added first

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the genetic code

• What is the nature of the genetic code?
• 1, nonoverlapping
• 2,devoiding of gaps or commas
• 3,three-base codons

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• The Elongation Mechanism

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Overview of Elongation(figure18.10)
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A Two-Site Model of the Ribosome

• The antibiotic puromycin(????) It looks like an
  aminoacyl-tRNA. So , it can bind to the A site of
  a ribosome and form a peptidyl bond with the
  peptide in the P site , yielding a peptidyl
  puromycin which is not tightly bound to the
  ribosome and is soon released , aborting
  translation prematurely(figure18.12)

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The link between puromycin and the two-site model

• 1.Before translocation, because the A site is
  occupied by a peptidyl-tRNA , puromycin cannot
  bind and release the peptide
• 2.After translocation,the A site is open .So ,
  puromycin can bind and release the peptide.
• This defines two sites on the ribosomepuromycin
  reactive site (P),and a puromycin unreactive
  site(A)

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Puromycin structure and activity(18.12)
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Evidence of fMet-tRNA binding to the P site
(figure18.13)

• Results
• Panel a and c Met and fMet attached to tRNAf
  went to the P site and was released
• b Met attached to tRNAm stayed in the A site
  and not released by puromycin

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Elongation step1Binding an Aminoacyl-tRNA to
the A Site of the Ribosome

• Three elongation factors
• EF-Tu EF-Ts EF-G
• These three factors participate in the first and
  third steps in elongation.

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Effects of EF-T and GTP on phe-tRNA binding to
ribosomes and on poly-phe synthesis(figure18.14)

• The EF-T-dependent binding to ribosome required
  GTP and considerable nonenzymatic binding
  occurred in the absence of EF-T and GTP
• Polymerization requires both EF-T and EF-G and a
  high concentration of GTP

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Mode of binding aminoacyl-tRNA to the ribosome A
site(figure18.15)
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Three evidences for the formation of the ternary
complex (AA-tRNAEF-Tu--GTP)

• First experiment
• Scientists found EF-T preparation and GTP could
  form a complex that can be retained by a
  nitrocellulose (????) filter
• However,when adding an aminoacyl-tRNA to the
  EF-Tu-GTP complex, the complex was released from
  the filter
• Hypothesisthe ternary complex could no longer
  bind to the filter.

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Evidence1Effect of aminoacyl-tRNA and deacylated
tRNA on the EF-Tu-GTP complex (figure18.16)

• ResultPhe-tRNA cause a big decrease in the
  amount of complex bound,while deacylated tRNA
  had little effect

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Evidence2Appearance of EF-T in nitrocellulose
filtrate(??) after addition of aminoacyl-tRNA

• Result only when EF-T , phe-tRNA,and GTP were
  all present did an EF-T complex pass through the
  filter
• (figure18.17)

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Evidence 3(figure18.18)

• Sephadex G100this gel filtration resin(??)
  excludes relatively large protein ,such as
  EF-T,So they flow through rapidly
• By contrast,relatively small substances like GTP
  ,and even phe-tRNA enter the pores in the resin
  and are thereby retarded,So they flow through
  slowly

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Formation of a ternary complex among EF-T ,
aminoacyl-tRNA ,and GTP (figure18.18)

• Resultboth GTP and phe-tRNA were found in a
  large-molecule fraction(20),so they were bound to
  the EF-T in a complex

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Effect of EF-Ts on ternary complex
formation(figure18.19)

• Result EF-Ts stimulates complex formation only
  when EF-Tu-GDP was the substrate. EF-Tu-GTP and
  EF-Tu plus GTP could form the complex
  spontaneously , with no help from EF-Ts

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• How does EF-Ts perform its exchange duty?

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Displacement of GDP from an EF-Tu-GDP complex by
EF-Ts(figure18.20)
AEF-Ts500 units BEF-Ts14000 units CEF-Ts
25000 units Result the more EF-Ts , the
little GDP in the complex
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• Elongation Step 2
• Peptide bond formation

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The puromycin reaction as an assay for peptidyl
transferase(Figure18.22)

• (a) standard puromycin reaction
  (b)Reaction with 50S only
• Result The ribosome itself , working as
  peptidyl transferase, forms peptide bonds

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• How to distinguish the released peptidyl-tRNA
  from the peptidyl-tRNA still bound to ribosomes?

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Puromycin assay for peptide bond
formation(Figure18.23)

• (a)a negative controlwith no puromycin ,as a
  result,the poly(phe) remained bound to the 50s
  subunit
• (b)a positive controlwith puromycin and treated
  with urea (??)and RNase. poly(phe) was
  released

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• What part of the 50S subunit had peptidyl
  transferase activity?

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Effects of protein-removing reagents(??) on
peptidyl transferase activities(Figure18.24)

• Lanes 1-4E-coli
• Lanes5 -9Thermus aquaticus
• (A)peptidyl transferase activity of E 50S
  survived SDS and PK but not phenol(??)
• (B) peptidyl transferase activity of
• T 50S survived all three

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Sensitivity of Thermus aquaticus fragment
reaction to peptidyl transferase inhibitors and
RNase (Figure18.25)

• ResultThe fragment reaction carried out by
  Thermus aquaticus 50S subunits are inhibited by
  carbomycin(???), chloramphenical(???), and RNase

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• From the two experiments above,We can conclude
• Ribosomal RNA is the component of peptidyl
  transferase

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Elongation Step 3Translocation

• Each translocation event moves the mRNA one
  codons length , 3nt, through the ribosome ,GTP
  and EF-G are necessary for translocation

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Important knowledge

• 1. Messenger RNAs are read in the 5-3direction
  ,the same direction in which they are synthesized
  .Proteins are made in the amino-carboxyl
  direction
• 2.The genetic code is a set of three-base codons
  and it is nonoverlapping ,devoiding of gaps or
  commas

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• 3.Three steps of elongation and the function of
  puromycin in defining A site and P site of the
  ribosome
• 4.The functions of three elongation factors
  EF-Tu EF-Ts EF-G and how EF-Ts regenerates an
  EF-Tu-GTP complex

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• 5.Peptide bond are formed by a ribosomal enzyme
  called peptidyl transferase , which resides on
  the 50s ribosomal particle . Ribosomal RNA is the
  component of peptidyl transferase

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Important experiments

• Figures
• 18.1 18.2 18.13 18.14
  18.16 18.17 18.18 18.19 18.20
  18.23 18.24 18.25

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• Thank you