Loop | Gayathri Silva

H. pylori is missg the glutamyl- and asparagyl-tRNA synthetas (GlnRS and AsnRS, rpectively). Consequently, H. pylori an direct amoacylatn pathway to generate Gln-tRNAGln and Asn-tRNAAsn. Wh this procs, Asn-tRNAAsn is produced by misacylatn of tRNAAsn wh aspartate by a non-discrimatg aspartyl-tRNA synthetase (ND-AspRS). Next, the heterotrimeric, glutame-pennt amidotransferase (lled AdT or GatCAB) nverts the misacylated Asp-tRNAAsn to Asn-tRNAAsn. A parallel pathway exists for the synthis of Gln-tRNAGln, where misacylatn of tRNAGln wh glutamate is talyzed by a tRNAGln-specific glutamyl-tRNA synthetase (GluRS2) to generate Glu-tRNAGln; this misacylated termediate is nverted to Gln-tRNAGln by the same AdT. This pennce on misacylated termediat for prote synthis suggts a requirement for addnal mechanisms to prevent the misrporatn of Glu and Asp to protes place of Gln and Asn. H. pylori elongatn factor (EF-Tu) provis one such machery, but is not sufficient to mata translatnal accuracy, suggtg the need for addnal mechanisms. Hp0495 and Hp0100 were intified by yeast two-hybrid (Y2H) as potential new players tRNA amoacylatn and fily. By Y2H, Hp0495 showed teractns wh EF-Tu and Hp0100 was nnected to both ND-AspRS and AdT. The work prented this dissertatn examed the possible rol of both of the protes direct amoacylatn and promotg tRNA accuracy. Usg SPR, native gels, and size excln chromatography, we have shown that Hp0495 forms plex wh EF-Tu both s GDP and GTP forms, but preferentially bds EF-Tu*GTP. In llaboratn wh a lleague, Dr. Keng-Mg Chang, we have disvered that Hp0495 bds ATP, glutamate, and eher tRNAGlu1 or tRNAGln and forms tRNAGln-pennt plex wh GluRS2. Also, Hp0495 bds acylated tRNAs more tightly than amoacylated tRNAs for the five s tted this work. Hp0495 has siari to ACT/RAM domas; the domas typilly regulate different aspects of amo acid and nucleoti metabolism. Our hypothis is that Hp0495 might be a regulatory prote, sensg tRNAGlu1/tRNAGln and directg one or both to eher GluRS1/GluRS2 for amoacylatn or for functns outsi prote translatn. The hp0495 gene is an operon wh mraY and murD, the two enzym are volved cell wall bsynthis, suggtg that Hp0495 might be volved regulatg this procs. There is also precence for the volvement of EF-Tu cell wall bsynthis, supportg this hypothis. In llaboratn wh a lleague, Dr. Gayathri Silva, we have monstrated that Hp0100 is, fact, an sential ponent of a tRNA-pennt Asn-transamidosome plex. This plex ntas