Nucleoside Phosphoramidate Monoesters: Potential Pronucleotides

1
tRNA Activation (charging) by aminoacyl tRNA
synthetases
Aminoacyl tRNA synthetase

• Two important functions
• Implement genetic code
• Activate amino acids for
• peptide bond formation
• The key enzymes
• Amanoacyl-tRNA synthetases

2
Aminoacyl-tRNA Synthesis
Summary of 2-step reaction 1. amino acid ATP ?
aminoacyl-AMP PPi 2. aminoacyl-AMP tRNA ?
aminoacyl-tRNA AMP The 2-step reaction is
spontaneous overall, because concentration of PPi
is kept low by its hydrolysis, catalyzed by
Pyrophosphatase.
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tRNA Activation by aminoacyl tRNA synthetases
1. Aminoacyl-AMP formation
HO
O
R
(-)O
P
O
O(-)
O
O
O
P
Adenine

PPi
C
P
R
O
O
O
O-
O
Adenine
O
O(-)
P
O
C
O
O-
O
OH
OH
2Pi
OH
OH
2. Aminoacyl transfer to the appropriate tRNA
R
R
O
O
O
Adenine
HO-ACC-tRNA
ACC-tRNA

AMP

C
C
P
O
O
O-
O
O
OH
OH
Overall reaction amino acid tRNA ATP ?
aminoacyl-tRNA AMP PPi
4
Classes of Aminoacyl-tRNA Synthetases
Class I Arg, Cys, Gln, Glu, Ile, Leu, Met,
Trp, Tyr, Val (Generally the Larger Amino
Acids)

• Class II Ala, Asn, Asp, Gly, His , Lys, Phe,
  Ser, Pro, Thr
• (Generally the smaller amino acids)
• Main Differences between the two classes
• Structural differences. Class I are mostly
  monomeric,
• class II are dimeric.
• Bind to different faces of the tRNA molecule
• 3. While class I acylate the 2 hydroxyl of the
  terminal Ado,
• class II synthetases acylate the 3-OH

5
Class I and II synthetases bind to different
faces of the tRNA molecule
6
Class I synthetases acylate the 2-OH
Class II synthetases acylate the 3-OH
7

• The accuracy of protein synthesis depends on
  correct
• charging of tRNAs with amino acids
• tRNA synthetases must link tRNAs with their
  correct amino
• acids.
• 2. tRNA synthetases recognize correct amino acids
  by specific
• binding to the active site and proofreading.
• 3. tRNA synthetases recognize correct tRNAs via
  by interacting with
• specific regions of tRNA sequence.

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• The accuracy of protein synthesis depends on
  correct
• charging of tRNAs with amino acids
• tRNA synthetases must link tRNAs with their
  correct amino
• acids.
• 2. tRNA synthetases recognize correct amino acids
  by specific
• binding to the active site and proofreading.
• 3. tRNA synthetases recognize correct tRNAs via
  by specific
• regions of tRNA sequence.

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The acylation site of threonyl tRNA synthetase
contains a Zinc ion that interacts with the OH
group of Threonine
10
Some amino acids have the same functional groups
and differ only by size
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tRNA Synthetase Proofreading

• Double sieve based on size
• Flexibility of the acceptor stem essential

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Isoleucil-tRNA Synthetase Proofreading based on
size
O
O
O
tRNAIle
O
tRNAIle
O
O
tRNAIle
O
tRNAIle
O
Val
Ile
Misacylation
Correct Acylation
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Valyl tRNAVal Synthetase Proofreading
hydrophobic/polar recognition motif
OH
O
O
tRNAVal
O
O
tRNAVal
Difference in Hydrophobicity
HO
O
O
tRNAVal
O
tRNAVal
O
Val
Thr
Correct Acylation
Misacylation
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• The accuracy of protein synthesis depends on
  correct
• charging of tRNAs with amino acids
• tRNA synthetases must link tRNAs with their
  correct amino
• acids.
• 2. tRNA synthetases recognize correct amino acids
  by specific
• binding to the active site and proofreading.
• 3. tRNA synthetases recognize correct tRNAs via
  using specific
• regions of the tRNA sequence.

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tRNA Recognition by Synthetases

• different recognition motif depending on
  synthetase
• usually just a few bases are involved in
  recognition
• Can involve specific recognition of the anticodon
  (e.g. tRNAMet), stem sequences can (e.g.
  tRNAAla), both stem regions and anticodon (e.g.
  tRNAGln), or, less frequently, D loop or T loop
  bases.

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Secondary Structure of Transfer RNA molecule
60-93 nt long
7 bp acceptor stem
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Examples of tRNA Recognition by aminoacyl tRNA
Synthetases
tRNAAla
tRNAPhe
tRNASer
3'
OH
OH
3'
3'
OH
P
5'
P
5'
P
5'
U70
G3
C11
A
G24
D
G34
A36
A35
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Threonyl tRNA synthase complex with tRNA
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Codon-anticodon recognition between tRNA and mRNA
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The relationship between the number of codons,
tRNAs, and synthetases
Total of 61 codons, but not 61 tRNAs! The same
tRNA can recognize more than one
codon Example Codon tRNA Synthetase GCU GCC
tRNAAla (5-IGC-3) alanyl tRNA synthetase GCA

5
3
CGI anticodon 5-GCU (C,A)-3 codon
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Genetic Code
22
Codon Anticodon Recognition

• The first two interactions (XY-XY) obey
  Watson-Crick
• base pairing rules.
• 2. The third interaction (ZZ) is less strict
  (Wobble pairing is allowed)

3 2 1
t RNA- 3'-X Y Z -5' anticodon mRNA- 5'-XYZ-3'
codon
1 2 3
The Third Base of Codon is Variable
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Wobble base pairing rules
first anticodon base (Z) third codon base
(Z) C G A U U A or G G C or
U I U, C, or A
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tRNA Anticodon-Codon Recognition
Adenosine
Guanosine
Inosine
O
O
N
N
N
HN
N
HN
N
N
HN
N
N
Ribose
tRNAAla
5'
5'
5'
C
G
I
C
G
I
C
G
I
Anticodon
3'
3'
3'
Codon
G
C
C
G
C
A
G
C
U
3'
5'
3'
5'
5'
3'
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tRNA Anticodon-Codon Recognition
5'
5'
5'
C
G
I
C
G
I
C
G
I
Anticodon
3'
3'
3'
Codon
G
C
U
G
C
C
G
C
A
3'
5'
3'
5'
5'
3'
5'
5'
C
G
G
C
G
G
Anticodon
3'
3'
Codon
G
C
U
G
C
C
3'
5'
5'
3'
5'
5'
C
G
U
C
G
U
Anticodon
3'
3'
Codon
G
C
A
G
C
G
3'
5'
5'
3'
5'
5'
C
G
C
C
G
A
Anticodon
3'
3'
Codon
G
C
G
G
C
U
3'
5'
5'
3'
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Genetic Code
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Overview of Protein Synthesis Take Home Message
1) Translation of the genetic code is dependent
on three base words that correspond to a single
amino acid. 2) The mRNA message is read by tRNA
through the use of a three base complement to the
three base word. 3) A specific amino acid is
conjugated to a specific tRNA (three base
word). 4) Amino acid side chain size,
hydrophobicity and polarity govern the ability of
tRNA synthetases to conjugate a specific three
base message with a specific amino acid.