Structure-Function Studies Of Tryptophanyl-tRNA Synthetase

Accurate translation of genetic information into proteins is of vital importance of all living organisms. Aminoacyl-tRNA synthetases (aaRS) play a crucial role in the quality control of protein synthesis through selection and transfer of the correct amino acid to its cognate tRNA. Diversities occurred between TrpRS or tRNATrp from different origins during evolution. This report studied the structure-function of human TrpRS and Bacillus subtilis TrpRS focusing on their differences.Two residues, K149 and E153 in B. subtilis TrpRS exhibit phylogenic diversity in sequence alignment. Mutation at the two sites markedly affected catalytic activities at both steps, suggesting their importance to enzyme function. Exchanging side chain electric charge of the two residues from prokaryotic mode to eukaryotic mode resulted in a dramatic drop in aminoacylation of prokaryotic tRNA but an obvious increase in the aminoacylation of eukaryotic tRNA. This suggests these two sites may specifically recognize the species-specific elements in tRNA.Despite their similar overall structure, prokaryotic TrpRS and eukaryotic TrpRS have different carboxyl terminal structure arrangement. Sequential deletion and site mutations revealed that B. subtilis TrpRS carboxyl terminus contribute greatly to both tryptophan activation and tRNA esterification. R327 and K328 are key residues in the carboxyl terminus. Change at these sites affected not only the amino acid activation process, but also the aminoacylation reaction. Mutant enzyme affinity to substrate tryptophan changed dramatically in the first step. Further studies using tRNA minihelixes suggested the involvement in cross subunit communication of carboxyl terminus upon tRNA binding. Compared with its prokaryotic counterparts, human TrpRS has an extra NH2 terminal peptide of about 140 residues. The enzyme remains active when 80 residues were deleted from this extra peptide, the mutant enzyme is even more active than wild type enzyme. The enzyme lost its activities when 94 residues were deleted, which suggests the indispensable role of the peptide from residue 81 to 94. Sequence alignment revealed this peptide is very conservative through TrpRSs of Archea and eukaryotic origin. Trp88 and Pro87 in this peptide were identical in all the TrpRS aligned, suggesting their importance in function. Structure analysis showed a smallβ-hairpin formed by this peptide may participate in the tryptophan activation step. Point mutation further confirmed that Trp88 is key residue in this structure. Interestingly, the inactive mutant N94d and W88A could tryptophanylate bovine tRNATrp at a higher level when prokaryotic TrpRS was added to the reaction system, suggesting that the acyl-transfer activity was not impaired as much as tryptophan activation ability was done. The observation that N94d and W88A could utilize intermediate product Trp-AMP formed by B. subtilis TrpRS the relative independence of the two steps in aminoacylation reaction.