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.