1. Peter John M.Phil, PhD Atta-ur-Rahman School of Applied Biosciences (ASAB) National University of Sciences & Technology (NUST)

2. tRNAs are charged with amino acids by synthetases
Aminoacyle-tRNA synthetases are enzymes that charge tRNA with an amino acid to generate aminoacyle-tRNA in a two-stage reaction that uses energy by from ATP
There are 20 aminoacyle-tRNA synthetases in each cell.
Recognition of a tRNA is based on a small number of point of contact in the tRNA seq

3. Aminoacyle synthetase Charged tRNA
All synthetases function in two steps
(i) Amino Acid react with ATP to form Aminoacyle Adenylate
(ii) Activate Amino Acid transferred to tRNA releasing AMP

4. Aminoacyle-tRNA synthetases

5. Over all reaction Amino acid + ATP ↔ Aminoacyl-AMP + PPi
Then, the amino acid residue is transferred to the tRNA:
Aminoacyl-AMP + tRNA ↔ Aminoacyl-tRNA + AMP The net reaction is:
Amino acid + ATP + tRNA ↔ Aminoacyl-tRNA + AMP + PPi

6. Aminoacyle tRNA Reaction Mechanism
acyl-tRNA
Synthetase
Gly
Amino-
acyl-tRNA
Synthetase A P
Amino-
acyl-tRNA
Synthetase A P
Gly A P
ATP

7. Aminoacyle tRNA Reaction Mechanism
Gly P
Pyrophosphate A
ATP
Amino-
acyl-tRNA
Synthetase
AMP
CCA
Gly
CCA
Aminoacyl-
tRNA
Amino-
acyl-tRNA
Synthetase

8. Over all reaction

9. Isoaccepting tRNA
Multiple tRNAs representing the same codon are called “ Isoaccepting RNA” or “ cognate tRNA”
Accepter stem & anticodon stem make tight contact with synthetase & mutation in this region disrupt this contact
Isoaccepting tRNAs are charged by same/single Aminoacyle tRNA synthetase

10. Isoaccepting tRNA
All tRNAs are divided into groups & each group identified its particular synthetase
tRNAs are recognized by synthetase by small no of bases (1-5 bases)

11. tRNA-Synthetase Interaction
Three features are used in recognition
(i) At least one base of anticodon
(ii) Last pair in the acceptor stem
(iii) Discrimination b/w acceptor stem & CCA terminus
This is not the over all rule, so recognition is “Idiosyncratic” each following its own rule

12. Aminoacyle tRNA synthetases size & structure
Size of Aminoacyle tRNA synthetase is kDa & may be monomeric, dimeric/tetrameric
Aminoacyle tRNAs synthetase are divided into two groups, division is based on the structural domain that contain the active site.
Each group contain 10 enzyme

13. Aminoacyle tRNA sythetase Structure
Aminoacyle tRNA sythetase have the following Domains
(i) Catalytic Domain ATP & AA site (N-Treminal catalytic domin)
(ii) tRNA acceptor domain
(iii) anticodon binding domain
(iv) oligomerization domain

14. Aminoacyle tRNA synthetase Group Aminoacyle tRNA synthetase Class I & II
Class I AA tRNA synthetase have N-terminal catalytic domain identified by the presence of 2 short conserved seq called “ signature seq” these are mostly monomer/some homodimers
Class II mostly homodimers & 2 are tetramers

15. Aminoacyle tRNA synthetase Class I & II
Class I tRNA synthetase contact tRNA at the minor grooves of the stem & at the anti codon
Class II aminoacyle tRNA synthetase contact tRNA at the major groove of the acceptor helix at the anti codon loop

16. Aminoacyle tRNA synthetase Class II
Class II aminoacyle tRNAs synthetase share general similarities of the seq in their catalytic domain
(i) Active site with antiparallel β-sheet surrounded by alpha helices
(ii) N-terminal anticodon domain
(iii) oligomerization domain widely open

17. Aminoacyle tRNA Synthetase

18. Proof Reading by Synthetase to improve accuracy
Specificity of recognition of both amino acid and tRNA is controlled by aminoacyle-tRNA synthetases by proofreading reactions that reverse the catalytic reaction if the wrong component has been incorporated.
Each synthetase distinguished one out of 20 AAs & must differentiate cognate tRNAs from the total set

19. Proof Reading by Synthetase
Two ways by which synthetases recognize their substrate
(i) The affinity of binding is sufficient to control the entry of substrate. Only correct amino acid & tRNA form a stable attachment to the site
(ii) After some stage of reaction the incorrect substrate is dissociated which is called “Proof Reading”

20. tRNA binding to synthetase
tRNA binding to synthetase in two stages
(i) correct tRNA binds & conformational change occur in the tRNA,
(ii) incorrect tRNA binds, no conformational change & it is dissociated which is called kinetic proof reading

21. Chemical Proof Reading
Incorrect AA is transformed into another form/hydrolyzed
(i) Met Homo cysteine thiolactone
(By product in E. Coli)
(ii) Incorrect AA Hydrolyzed

22. Kinetic Proof Reading

23. Chemical Proof Reading
When Incorrect amino acid added
(i) Conformational change in aminoacyle-tRNA
(ii) Hydrolysis of aminoacyle-tRNA

24. Chemical Proof Reading

25. Size Discrimination
Ile-tRNA synthetase have two site & use AA size for its discrimination
(i) Synthetic/Active site
(ii) Editing/Hydrolytic site
Synthetic site is too small if AA enter this, it is blocked by editing site

26. Double Sieve model

27. Thanks