Protein Synthesis In depth

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Protein Synthesis In depth

• Chapter 29

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No loss in Translation

• mRNA to Protein Translation
• More complex than replication or transcription
• Highly conserved in all life forms (this chapter
  focuses on prokaryotes)
• High speed 20 aa/sec
• Low error frequency required

Observed value of error (many large proteins are
multi-subunit)
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Basic Translation Reaction

• Amino acid at 3 end of specific tRNA
• Activation of tRNA catalyzed by aminoacyl-tRNA
  synthetase
• ATP cleavage drives tRNA activation reaction
  (remember thermodynamics must be satisfied)

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tRNA Molecules

• General features
• 73 to 93 ribonucleotides (25 kD)
• Methylated or dimethylated versions of bases
  (structure)
• Cloverleaf structure with anti-codon loop
• 5 phosphorylated
• Amino acid at 3

• IGC complementary to GCC (A)

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3-D structure

• L-shaped molecule anti-codon and amino acid
  attachment at opposite ends
• CCA conserved sequence for amino acid
  attachment
• 4 helices

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Activation of tRNAs

• Fidelity of the attachment of amino acids to a
  tRNA molecule is critical
• Amino acids first activated by adenylation
• Then transferred to tRNA
• Hydrolysis of PPi drives reaction forward
• Equivalent of 2 ATP consumed for each
  aminoacyl-tRNA
• Activation and transfer of particular amino acid
  are catalyzed by the same aminoacyl-tRNA
  synthetase.

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Enzyme specificity How do synthetases recognize
right tRNA?

• Recognize anti-codon loops and acceptor stems of
  tRNA
• Threonyl-tRNA synthetase example
• How does enzyme discriminate between Ser and Val?

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• Zn ion in binding site coordinate bonds with 2
  His and 1 Cys Thr OH and amine groups
  coordinately bonds also Asp also interacts with
  OH
• Val methyl group can not bond Ser can be
  incorporated at about 1/100 1/1000 times that
  of Thr

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Proofreading additional fidelity

• In vitro synthesis of Ser-tRNAThr, then incubate
  with Threonyl-tRNA synthetase
• Rapid hydrolysis of Ser to release free tRNAThr
• Acylation site rejects larger amino acids via
  steric considerations
• Editing sites use hydrolysis to remove smaller
  amino acids
• 1/10,000 error frequencies achieved by this
  combination of proof-reading and steric
  considerations

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How do Synthetases recognize correct tRNA?

• Aminoacyl tRNA synthetases are the only molecules
  in biology that know the genetic code
• Combination of interactions
• Anticodon loop (some more than others)
• Acceptor stem
• Other regions (glutaminyl tRNA synthetase)

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Threonyl-tRNA synthetase complex
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Glutaminyl-tRNA synthetase complex
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Ribosomes molecular machines

• Coordinate charged tRNAs, mRNA, and nascent
  polypeptides during protein synthesis
• Macromolecular complex
• 50S subunit 34 proteins 2 RNA molecules
  L1-L34 23S and 5S RNA
• 30S subunit 21 proteins 1 RNA S1-S21 16S
  RNA
• S20 and L26 are identical
• One copy of each RNA, two copies L7 and L12, 1
  copy of all other proteins
• L7 and L12 identical except L7 N-term is
  acetylated

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16S rRNA from E. coli
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Protein Synthesis

• Amino to carboxyl direction
• mRNA translated 5 to 3 direction
• Transcription is also 5 to 3
• So partially transcribed mRNA can be translated
• In prokaryotes, transcription and translation are
  closely coupled in space and time

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• Prokaryotes - Many ribosomes can translate a
  single mRNA at the same time
• Polysome or Polyribosome

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Start Signals

• Usually about 25 bases from 5 terminus
• Prokaryote mRNAs also polycistronic multiple
  proteins from single mRNA molecule
• Initiation sites
• Shine-Dalgarno sequence AUG initiator codon

Shine-Dalgarno sequence complimentary binding
to rRNA
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Protein synthesis initiation

• E. coli - Formylmethionyl tRNA
• Removed from protein after about 10 amino acids
  in 50 of proteins
• Specific mechanism for f-Met synthesis and
  incorporation

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Ribosome tRNA binding Sites

• A site aminoacyl
• P site peptidyl
• E site - exit

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Tunnel for growing polypeptide chain
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Why Formylation of Met?
Termination of translation if not formylated!
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Animated versions

• Transcription Animations
• Narrated Global View Animation of Protein
  synthesis Transcription
• Silent Animation Narrated Animation