tRNA Activation (charging) by aminoacyl tRNA synthetases
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Transcript of tRNA Activation (charging) by aminoacyl tRNA synthetases
tRNA Activation (charging) by aminoacyl tRNA synthetases
AminoacyltRNA synthetase
Two important functions:
1. Implement genetic code
2. Activate amino acids forpeptide 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
C+H3N
R
O
O(-) PO
O
OH OH
O O-
AdenineO
PO P
O(-)
HO O
O
(-)O
C+H3N
R
O
OP
O
O
OH OH
O O-
Aminoacyl adenylate (Aminoacyl-AMP)
+ PPiAdenine
1. Aminoacyl-AMP formation:
2. Aminoacyl transfer to the appropriate tRNA:
C+H3N
R
O
OAMP+ACC-tRNAC+H3N
R
O
OP
O
O
OH OH
O O-
HO-ACC-tRNAAdenine +
2Pi
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
O
N
NN
N
NH2
O
O
HH
HH
PO
O
tRNA
CO
CH
R
NH3
OH
o-
Class I synthetasesacylate the 2’-OH
O
N
NN
N
NH2
O
OH
HH
HH
PO
O
tRNA
CO
CH
R
NH3
O
o-
Class II synthetasesacylate the 3’-OH
The accuracy of protein synthesis depends on correctcharging of tRNAs with amino acids
1. tRNA synthetases must link tRNAs with their correct aminoacids.
2. tRNA synthetases recognize correct amino acids by specificbinding to the active site and proofreading.
3. tRNA synthetases recognize correct tRNAs via by interacting withspecific regions of tRNA sequence.
The accuracy of protein synthesis depends on correctcharging of tRNAs with amino acids
1. tRNA synthetases must link tRNAs with their correct aminoacids.
2. tRNA synthetases recognize correct amino acids by specificbinding 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
H2N CH C
CH
OH
O
OH
CH3
H2N CH C
CH
OH
O
CH3
CH3
Thr Val
H2N CH C OH
O
H2N CH C
CH
OH
O
CH3
CH3
Ile Val
CH CH3
H2CCH3
Some amino acids have the same functional groups and differ only by size:
tRNA Synthetase Proofreading
•“Double sieve” based on size• Flexibility of the acceptor stem essential
Isoleucil-tRNA Synthetase: Proofreading based on size
CH3
O+H3N
tRNAIleO
CH3
Smaller Hydrolytic Site
Larger Acylation Site
CH3H3C
ONH3+
O
Larger Acylation Site
Smaller Hydrolytic Site
tRNAIle
CH3
O+H3N
tRNAIleO
CH3
Correct Acylation
H3C CH3
O+H3N
tRNAIleO
Misacylation
Difference in Size
Ile Val
Valyl tRNAVal Synthetase Proofreading:
hydrophobic/polar recognition motif
3HC CH3
O+H3N
tRNAValO
Hydrophobic Acylation Site
Polar Hydrolytic Site
CH3 CH3
O+H3N
tRNAValO
Correct Acylation
HO CH3
O+H3N
tRNAValO
Misacylation
OHH3C
ONH3+
OtRNAVal
Polar Hydrolytic Site
Hydrophobic Acylation Site
Difference in Hydrophobicity
Val Thr
The accuracy of protein synthesis depends on correctcharging of tRNAs with amino acids
1. tRNA synthetases must link tRNAs with their correct aminoacids.
2. tRNA synthetases recognize correct amino acids by specificbinding 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
H2C NH
NH2C
O
O
dihydrouridine (UH2)
NHHN O
O
pseudouridine (
60-93 nt long
7 bp acceptor stem
A
OH
P5'
3'
U70G3
A C C
OH
P5'
3'A C C
OH
P5'
3'A C C
tRNAAla tRNAPhe
G34A35
A36
Examples of tRNA Recognition by aminoacyl tRNA Synthetases
tRNASer
C11
G24
D
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 Synthetase
GCUGCC tRNAAla (5’-IGC-3’) alanyl tRNA synthetaseGCA
CGI anticodon
5’-GCU (C,A)-3’ codon
5’3’
Genetic Code
Codon : Anticodon Recognition
t RNA- 3'-X Y Z -5' anticodonmRNA- 5'-X’Y’Z’-3' codon
1 2 3
3 2 1
The Third Base of Codon is Variable
1. The first two interactions (XY-X’Y’) obey Watson-Crickbase pairing rules.
2. The third interaction (ZZ’) is less strict (“Wobble” pairing is allowed)
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
t RNA- 3'-X Y Z -5' anticodonmRNA- 5'-X’Y’Z’-3' codon
1 2 3
3 2 1
tRNA Anticodon-Codon Recognition
HN
N N
N
O
Inosine
Ribose
N
N N H
N
NH2
HN
HN
N N H
N
O
Adenosine Guanosine
C G IG C C
C G IG C A
C G IG C UCodon
Anticodon 5'3'3'5'
5'3'3'5'5' 3'
3' 5'
NHN
N N
O
C1'
N
N
NH2
OC1'
C-I base pair
NN
NHN
NH2
NHN
N N
O
C1'A-I base pair
C1'
NH
N
O
O
N
HN
NN
O
C1'C1'
U-I base pair
tRNAAla
tRNA Anticodon-Codon Recognition
C G IG C U
C G IG C C
C G IG C ACodon
Anticodon 5'3'3'5'
5'3'3'5'5' 3'
3' 5'
C G GG C U
C G GG C CCodon
Anticodon 5'3'3'5'5' 3'
3' 5'
C G UG C A
C G UG C GCodon
Anticodon 5'3'3'5'5' 3'
3' 5'
C G CG C G
C G AG C UCodon
Anticodon 5'3'3'5'5' 3'
3' 5'
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.