Chapter 22 Protein Synthesis

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Chapter 22 - Protein Synthesis
mRNA
The ribosome, a complex of RNA and protein, is
the site where genetic information is translated
into protein
protein
Exam Tues Dec 1
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The Genetic Code
DNA
Transcription
RNA
5-CAGGGUGGUGAUAUGAAACCA------AUCGCUUGA-3
A specific protein e.g. insulin
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The Genetic Code
How is the info translated from NA to protein ?

• Codons - three letter genetic code
  (nonoverlapping)
• tRNA - adapters between mRNA and proteins
• Reading frame - each potential starting point for
  interpreting the 3 letter code

• DNA only four bases (A,T,G,C)
• Must code for 20 amino acids
• Two-base code 42 16 combinations
• Four-base code 44 256 combinations
• Three-base code 43 64 combinations

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Overlapping vs nonoverlapping reading of the
three-letter code
What is one benefit ?
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Three reading frames of mRNA

• Translation of the correct message requires
  selection of the correct reading frame

tRNA reads each codon
Key decide where to start
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Standard genetic code
(1962 1965, Khorana, Nirenberg in separate
laboratories)
(5-UUU-3)
(5-UUC-3)
(START)
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Features of the genetic code

• The genetic code is unambiguous. Each codon
  corresponds to only one amino acid.
• 2. There are multiple codons for most amino
  acids (code is degenerate)

3. The first two nucleotides of a codon are often
enough to specify a given amino acid (Gly
GG_ )

• 4. Codons with similar sequences specify similar
  amino acids
• 5. Only 61 of the 64 codons specify amino acids
• Termination (stop codons) UAA, UGA, UAG
• Initiation codon - Methionine codon (AUG)
  specifies initiation site for protein synthesis

Asp and Glu GU_ single mutation chem
similar AA
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Crack the genetic code
RNA Tie Club
20 members
16 had a specific amino acid
George Gamow (ALA)
4 members had specific nucleotide
F. Crick (TYR)
J. Watson (PRO)
Richard Feynman (GLY)
Combination of hard drinking and science
Melvin Calvin (HIS)
Edward Teller (LEU)
Leslie Orgel (THR)
Max Delbruck (TRY)
Sydney Brenner (VAL)
Erwin Chargaff (LYS)
1st meeting Woods Hole 1954
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Crack the genetic code
RNA Tie Club
Aim Solve the riddle of RNA structure and
understand how it built proteins
George Gamow (ALA)
F. Crick (TYR)
J. Watson (PRO)
Richard Feynman (GLY)
Combination of hard drinking and science
Melvin Calvin (HIS)
Edward Teller (LEU)
Leslie Orgel (THR)
Max Delbruck (TRY)
Sydney Brenner (VAL)
Erwin Chargaff (LYS)
1st meeting Woods Hole 1954
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Crack the genetic code
F. Crick (TYR)
Adapter hypothesis
George Gamow (ALA)
Mathematics used to establish that
3 letter code would be enough to define all 20
amino acids
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Crack the genetic code
Nonmembers of the RNA Tie Club
Marshall Nirenberg
1961
Johann Matthaei
Cell free system
RNA template, ribosomes,
nucleotides, amino acids, ATP
UUUUUUUUUUUU
FFFF
CCCCCCCCCCCCCC
PPPP
Har Gobind Khorana
UCUCUCUCU
Ser,Leu,Ser,Leu
Robert Holley
Determined the structure of tRNA
1965
Nobel prize 1968
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• Cloverleaf structure of tRNA

Amino acid

• Every cell must contain at least 20 tRNA (one for
  every amino acid)
• Each tRNA must recognize at least one codon

• Watson-Crick base pairing (dashed lines)
• tRNA has an acceptor stem and four arms
• Conserved bases (gray)

Complementary to the codon
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(No Transcript)
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tRNA Anticodons Base-Pair with mRNA Codons

• tRNA molecules are named for the amino acid that
  they carry (e.g. tRNAPhe)

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5
tRNAPhe

• Base pairing between codon and anticodon is
  governed by rules of Watson-Crick

Wobble position
AAG
A-U G-C
5
UUC
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Variable position
mRNA
(Phe)
However, the 5 anticodon position has some
flexibility in base pairing (the wobble
position)
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3
5
tRNAPhe
Wobble position
AAG
5
UUC
3
Variable position
mRNA
(Phe)
Deamination of G I
5
UUU
3
(Phe)
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Base pairing at the wobble position

• Inosinate (I) often found at 5 wobble position
• I can form H bonds with A, C, or U
• Anticodon with I can recognize more than one
  synonymous codon

Some bacteria get by with only 31 tRNA (not 61)
Weaker H-bond speeds up prot. Syn.
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Codon-anticodon recognition

• Wobble allows some tRNA molecules to recognize
  more than one codon
• Isoacceptor tRNA molecules - different tRNA
  molecules that attach the same amino acids
• Isoacceptor tRNAs identified by Roman numerals or
  codons tRNAIAla, tRNAIIAla or tRNAGCGAla
• Bacteria have 30-60 different tRNAs
• Eukaryotes have up to 80 different tRNAs

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Protein synthesis translation
Aminoacylation of tRNA
Charging of tRNA
By Aminoacyl-tRNA Synthetases
Initiation Chain elongation Termination
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Structure of the Aminoacyl-tRNA Linkage
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Aminoacylation of tRNA
The genetic code is translated by two adaptors
The first is the aminoacyl-tRNA synthetase. The
linkage is through a high energy bond created
with ATP.
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High energy bond
The second adaptor is the tRNA itself whose
anticodon forms base pairs with the appropriate
mRNA codon. An error in either step causes wrong
aa in peptide chain.
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Protein Synthesis Proceeds by the Addition of an
AA to the C-terminus of a polypeptide
Peptidyl Transferase
mRNA
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5
Energy stored in aminoacyl-tRNA used in formation
of peptide bond
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Aminoacyl-tRNA Synthetases

• Aminoacyl-tRNA - amino acids are covalently
  attached to the 3 end of each tRNA molecule
  (named as alanyl-tRNAAla)
• Aminoacyl-tRNA synthetases catalyze reactions
• Most species have at least 20 different
  aminoacyl-tRNA synthetases
• (1 per amino acid)
• Each synthetase specific for a particular amino
  acid, but may recognize isoacceptor tRNAs

• Aminoacyl-tRNAs are high-energy molecules (the
  amino acid has been activated)
• The activation of an amino acid by aminoacyl-tRNA
  synthetase requires ATP

Amino acid tRNA ATP Aminoacyl-tRNA AMP
PPi
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Synthetase binds ATP and corect AA Based on
size/charge/hydrophobicity
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Synthetase selectively binds tRNA based
on Structural features Anticodon Acceptor stem
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Ester linkage
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Specificity of Aminoacyl-tRNA Synthetase

• Attachment of the correct amino acid to the
  corresponding tRNA is a critical step
• Synthetase binds ATP and the correct amino acid
  (based on size, charge, hydrophobicity)
• Synthetase then selectively binds specific tRNA
  molecule based on structural features, anticodon
  and acceptor stem

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Proofreading Activity of Aminoacyl-tRNA
Synthetases

• Some aa-tRNA synthetases can proofread
• Isoleucyl-tRNA synthetase may bind valine instead
  of isoleucine and form valyl-adenylate
• The valyl-adenylate is usually then hydrolyzed to
  valine and AMP so that valyl-tRNAIle does not
  form

1 in 100
1 in 10000
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Ribosomes
Protein synthesis by a complex composed of the
ribosome
Catalyzes peptide bond formation
accessory protein factors
Assist ribosome
mRNA
Carries info
charged tRNA molecules
Carries activated AA
Initiation complex assembles at first mRNA
codon, and disassembles at termination step
Ribosome moves 5 to 3 along mRNA
Peptide grows N to C direction
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Ribosomes
Coupled transcription and translation in E.coli
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Ribosomes Are Composed of Both rRNA and Protein
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Ribosomes
Large subunit
Small subunit
30S
50S
Nobel Prize in Chemistry 2009
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Ribosomes Contain Two Aminoacyl-tRNA Binding Sites

• Ribosome must align two charged tRNA molecules so
  that anticodons interact with correct codons of
  mRNA
• Aminoacylated ends of the tRNAs are positioned at
  the site of peptide bond formation
• Ribosome must hold both mRNA and growing
  polypeptide chain

30S
50S
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Mechanism of Translation
1. Initiation

• 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

2. Elongation
3. Termination
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Initiator tRNA

• First codon translated is usually AUG
• Each cell contains at least two methionyl-tRNAMet
  molecules which recognize AUG
• The initiator tRNA recognizes initiation codons

• Bacteria N-formylmethionyl-tRNAfMet
• (Eukaryotes methionyl-tRNAiMet)

Second tRNAMet recognizes only internal AUG
EF-Tu interaction (does not bind
formyl-tRNAfMet)
IF2 interacts with formyl-tRNAfMet
formylmethionine
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Shine-Dalgarno sequences in E. coli mRNA

• Ribosome-binding sites at the 5 end of mRNA for
  several E. coli proteins
• In prokaryotes, the 30S ribosome binds to a
  region of the
• mRNA (Shine-Dalgarno sequence) upstream of the
  initiation
• sequence
• S-D sequence binds to a complementary base
  sequence at the 3 end of the 16S rRNA

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• Complementary base pairing of S-D sequence