Protein Synthesis part II

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Protein Synthesis part II
Aminoacylation of tRNA
Initiation Chain elongation Termination
Ribosome moves 5 to 3 along mRNA
Peptide grows N to C direction
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Initiation of Translation

• The translation complex is assembled at the
  beginning of the mRNA coding sequence
• Complex consists of Ribosomal subunits mRNA
  template to be translated Initiator tRNA
  molecule Protein initiation factors

IF-1 IF-2 IF-3
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Initiation of Translation
Initiator tRNA molecule
Bacteria N-formylmethionyl-tRNAfMet
Second tRNAMet recognizes only internal AUG
Protein initiation factors
IF-1 IF-2 IF-3
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Formation of the prokaryotic 70S initiation factor
Inititation factors IF1, IF2, and IF3 are
required to form the ribosomal complex
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Translation Initiation in Eukaryotes

• Eukaryotic initiation factor 4 (eIF-4), (or cap
  binding protein, CBP) binds to the (5 end)
  7methylguanylate cap of eukaryotic mRNA
• A preinitiation complex forms (40S ribosome,
  aminoacylated initiator tRNA, other factors) and
  searches the mRNA 5 3 for an initiator codon
• The Met-tRNAiMet binds to AUG, and the 60S
  ribosomal subunit binds to complete the complex

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Chain Elongation is a Three-Step Microcycle

• The initiator tRNA is in the P site
• Site A is ready to receive an aminoacyl-tRNA
• Elongation is a three-step cycle
• (1) Positioning the correct aa-tRNA in site A
  (2) Formation of a peptide bond (3)
  Shifting mRNA by one codon

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Translating an mRNA Molecule--Elongation
three-step cycle
Positioning the correct aa-tRNA in site A
Formation of a peptide bond
Shifting mRNA by one codon
Translocation
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Positioning of the aminoacyl-tRNA
Insertion of aa-tRNA by EF-Tu during chain
elongation
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(No Transcript)
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Cycling of EF-Tu-GTP
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Peptidyl Transferase Catalyzes Peptide Bond
Formation

• Substrate binding site in 23S rRNA and 50S
  ribosomal proteins
• Catalytic activity from 23S rRNA (an
  RNA-catalyzed reaction)

Adenine abstracts proton
donates proton

• Peptidyl transferase activity is contained within
  the large ribosomal subunit

Catalytic activity from 23S rRNA (an
RNA-catalyzed reaction)
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A Pocket in the 23S Ribosomal RNA is the Catalyst
for the Peptidyl Transferase
Activity
N3 of the Adenine in the catalytic pocket of 23S
rRNA abstracts a proton from the aa acylated to
the tRNA. The Amino N of the aa acid then
attacks the carboxyl group of the peptide in the
P site.
The protonated Adenine donates its hydrogen to
the Oxygen linked to the tRNA thus releasing the
tRNA.
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tRNA originally attached to peptide in the P
site is released
New Amino Acid
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Translocation Moves the Ribosome by One Codon

• Translocation step the new peptidyl-tRNA is
  moved from the A site to the P site, while the
  mRNA shifts by one codon
• The deaminoacylated tRNA has shifted from the P
  site to the E site (exit site)

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EF-G-GTP
GTP hydrolysis causes large conformational change
that moves peptidyl tRNA to P site
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the mRNA shifts by one codon
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Translational Elongation in Prokaryotes
EF-Tu is the most abundant protein in E. coli
-- 6 of total protein. It is a G protein.
EF-Tu and other translational G-proteins
interact with a cleft in the 50s subunit that
acts as a GEF
Guanine exchange factor
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EF-G is another G protein and hydrolysis of
its GTP powers translocation.
P site
A/P hybrid site
A site
Hydroysis of GTP by EF-G causes large
conformational change that moves tRNA from the
A/P hybrid site to the P site.
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Formation of the peptide chain

• Growing peptide chain extends from the
  peptidyl-tRNA (P site) through a tunnel in the
  50S subunit
• Newly synthesized polypeptide does not begin to
  fold until it emerges from the tunnel
• Elongation in eukaryotes is similar to E. coli
  EF-1a - docks the aa-tRNA into A site EF-1b -
  recycles EF-1a EF-2 - carries out
  translocation

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Termination of Translation

• E. coli release factors RF-1, RF-2, RF-3
• Translocation positions one of three termination
  codons in A site UGA, UAG, UAA
• No tRNA molecules recognize these codons and
  protein synthesis stalls
• One of the release factors binds and causes
  hydrolysis of the peptidyl-tRNA to release the
  polypeptide chain

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Protein Synthesis is Energetically Expensive

• Four phosphoanhydride bonds are cleaved for each
  amino acid added to a polypeptide chain
• Amino acid activation Two P bonds
• ATP AMP 2 Pi
• Chain elongation Two P bonds
• 2 GTP 2 GDP 2 Pi

More energy then req for formation of single
peptide bond
Compensate for loss of entropy
Specific order of aa in peptide
Specific aa linked to tRNA
Specific tRNA/codon interaction
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The Genetic Code
DNA
Transcription
RNA
5-CAGGGUGGUGAUAUGAAACCA------AUCGCUUGA-3
A specific protein e.g. insulin
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Final quiz
Compare Group I and Group II RNA splicing. Give
two differences regarding the mechanism.