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

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

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

Aminoacyle-tRNA synthetases

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

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

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

Over all reaction

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

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)

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

Aminoacyle tRNA synthetases size & structure Size of Aminoacyle tRNA synthetase is 40-110 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

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

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

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

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

Aminoacyle tRNA Synthetase

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

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”

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

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

Kinetic Proof Reading

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

Chemical Proof Reading

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

Double Sieve model

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