Transcript Nucleoside Phosphoramidate Monoesters: Potential

tRNA Activation (charging) by aminoacyl tRNA
synthetases
Aminoacyl
tRNA synthetase
Two important functions:
1.
Implement genetic code
2. Activate amino acids for
peptide bond formation
The key enzymes:
Amanoacyl-tRNA synthetases
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.
tRNA Activation by aminoacyl tRNA synthetases
1. Aminoacyl-AMP formation:
HO O
(-)O
O
P
R
O
O(-)
O
P
C
O
O
+H 3 N
O
O(-)
+H 3 N
R
P
O-
Adenine
O
O
C
O
O
Adenine
O
P
O
O-
+
PPi
OH OH
Aminoacyl adenylate
(Aminoacyl-AMP)
OH OH
2Pi
2. Aminoacyl transfer to the appropriate tRNA:
R
R
O
+H 3 N
C
O
O
P
O
O-
Adenine
O
+
HO-ACC-tRNA
O
+H 3 N
C
ACC-tRNA
+
AMP
O
OH OH
Overall reaction: amino acid + tRNA + ATP  aminoacyl-tRNA + AMP + PPi
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:
1. Structural differences. Class I are mostly monomeric,
class II are dimeric.
2. 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
Class I and II synthetases bind to different faces of the tRNA molecule
Class I synthetases
acylate the 2’-OH
Class II synthetases
acylate the 3’-OH
NH2
NH2
tRNA
N
tRNA
N
N
N
O
O
o-
oO
N
N
P
O
N
P
O
O
O
O
H
H
H
H
H
H
OH
O
O
H
O
H
O
C
C
CH
CH
R
OH
NH3
R
NH3
N
The accuracy of protein synthesis depends on correct
charging of tRNAs with amino acids
1. 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.
The accuracy of protein synthesis depends on correct
charging of tRNAs with amino acids
1. 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.
The acylation site of threonyl tRNA synthetase contains a Zinc ion
that interacts with the OH group of Threonine
O
H2N
CH C
O
OH
H2N
CH C
CH OH
CH CH3
CH3
CH3
Thr
Val
OH
Some amino acids have the same functional
groups and differ only by size:
O
H2N
CH C
CH CH3
H2C
Ile
O
OH
H2N
CH C
CH CH3
CH3
CH3
Val
OH
tRNA Synthetase Proofreading
•“Double sieve” based on size
• Flexibility of the acceptor stem essential
Isoleucil-tRNA Synthetase: Proofreading based on size
Larger
Acylation Site
Larger
Acylation Site
Smaller
Hydrolytic Site
Smaller
Hydrolytic Site
CH 3
H3C
CH 3
CH 3
O
O
NH 3 +
+H 3 N
tRNAIle
O
CH 3
O
tRNAIle
Difference in Size
H 3C
CH 3
O
O
+H 3 N
+H 3 N
O
CH 3
O
tRNAIle
Ile
Correct Acylation
Val
Misacylation
tRNAIle
Valyl tRNAVal Synthetase Proofreading:
hydrophobic/polar recognition motif
Hydrophobic
Acylation Site
3 HC
Polar
Hydrolytic Site
Hydrophobic
Acylation Site
Polar
Hydrolytic Site
CH 3
H 3C
O
OH
O
+H 3 N
NH 3 +
O
tRNAVal
tRNAVal
O
Difference in Hydrophobicity
CH3
CH 3
HO
CH 3
O
O
+H 3 N
+H 3 N
O
tRNAVal
Val
Correct Acylation
O
tRNAVal
Thr
Misacylation
The accuracy of protein synthesis depends on correct
charging of tRNAs with amino acids
1. 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.
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.
Secondary Structure of Transfer RNA molecule
60-93 nt long
7 bp acceptor stem
O
O
H2C
H2C
NH
NH
N
O
dihydrouridine (UH2)
HN
O
pseudouridine (
Examples of tRNA Recognition by aminoacyl
tRNA Synthetases
tRNAAla
5'P
G3
3'OH
A
C
C
tRNAPhe
5'P
tRNASer
3'OH
A
C
C
5'P
U70
C11
A
G24
D
G34
A35
A36
3'OH
A
C
C
Threonyl tRNA synthase complex with tRNA
Codon-anticodon recognition between
tRNA and mRNA
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
GCU
GCC
GCA
tRNAAla (5’-IGC-3’) alanyl tRNA synthetase
3’
Synthetase
5’
CGI
5’-GCU (C,A)-3’
anticodon
codon
Genetic Code
Codon : Anticodon Recognition
1. The first two interactions (XY-X’Y’) 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'-X’Y’Z’-3' codon
1 2 3
The Third Base of Codon is Variable
Wobble base pairing rules
3 2 1
t RNA- 3'-X Y Z -5' anticodon
mRNA- 5'-X’Y’Z’-3' codon
1 2 3
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
tRNA Anticodon-Codon Recognition
Adenosine
Inosine
NH 2
O
O
N
N
N
HN
N
H
N
Guanosine
N
N
N
HN
HN
N
H
N
Ribose
tRNAAla
Anticodon
Codon
3'
5'
C
G
NH2
O
O
I
C
N
C-I base pair
5'
3'
3'
5'
C
G
NH2
G
C
N
C1'
C1'
N
5'
3'
3'
5'
N
A-I base pair
C
G
G
C
5'
3'
I
U
O
N
N HN
HN
I
A
O
N
N
N HN
N
C1'
G
C
NH
N
N
C1'
C1'
O
N
O HN
N
N
U-I base pair
C1'
tRNA Anticodon-Codon Recognition
Anticodon
Codon
3'
5'
C
G
G
C
5'
3'
I
U
3'
5'
C
G
Anticodon
Codon
3'
5'
C
G
G
C
G
U
5'
Anticodon
Codon
3'
5'
C
G
G
C
U
A
5'
Anticodon
Codon
3'
5'
C
G
G
C
C
G
5'
3'
3'
3'
G
C
I
C
5'
3'
3'
5'
C
G
G
C
G
C
5'
3'
5'
C
G
G
C
U
G
5'
3'
5'
C
G
G
C
A
U
5'
3'
5'
C
G
G
C
3'
3'
3'
I
A
5'
3'
Genetic Code
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.