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  • br Materials and methods br Results In order to


    Materials and methods
    Results In order to address the role played by the catalytic and the anticodon-binding domains of E. coli GluRS in cognate and non-cognate aminoacylation, the two domains (NGluRS: 1–314; CGluRS: 318–471) of E. coli were expressed and purified (Fig. 1b). Prior to assessing the aminoacylation properties, structural and cognate and non-cognate binding studies were performed on the isolated domains.
    Discussion For many bacterial species tRNAGln discrimination is an inherent function of GluRS. It was earlier shown that the anticodon-binding domain of GluRS is responsible for such discrimination [11], [12], [13]. However, despite the knowledge that identity elements in tRNAGlu and tRNAGln are present both in the anticodon loop and the acceptor arm [7], [8], [9], [10], there has been no experimental attempt to explore the contribution of the catalytic domain of GluRS in tRNAGln discrimination. How does GluRS accomplish tRNAGlu glutamylation and tRNAGln discrimination in terms of contributions from its two domains? A naturally truncated GluRS variant (YadB), homologous to the catalytic domain, is capable of activating L-Glu yet unable to deliver the activated Glu to either tRNAGlu or tRNAGln[24]. A number of studies on isolated catalytic domains of other aaRSs have proved to be useful in delineating the functions of the catalytic and the anticodon-binding domains of aaRSs [31], [32], [33]. Following this strategy we addressed the question of tRNA discrimination by studying the N-terminal catalytic domain (NGluRS; 1–314) and the C-terminal anticodon-binding domain (CGluRS; 318–471) of EcGluRS. Although binding of the cognate substrates (L-Glu and tRNAGlu) was comparable for NGluRS and GluRS, an attenuated kcat substantially diminished the glutamylation activity of NGluRS. tRNAGlu binding to CGluRS and GluRS were comparable. However, the addition of CGluRS to NGluRS did not significantly alter NGluRS activity. Our results show that the intact anticodon-binding domain in wild type EcGluRS affects transition state energetics (kcat effect) despite being distant from the catalytic site. Isolated catalytic domain of other aaRSs, like Bacillus stearothermophilus LysRS [31] and EcCysRS [32], also showed a much diminished catalytic activity towards their cognate aminoacylation reaction, resulting mainly from the destabilization of the transition state in the cognate amino Fesoterodine Fumarate australia activation step without affecting the ground state of substrate binding. In another report it was shown that besides being active towards cognate aminoacylation, a minimalist version of EcGlnRS was found to charge a non-cognate tRNATyr-derived amber suppressor (supF) with glutamine [33]. Surprisingly, even with a diminished activity and absence of the anticodon-binding domain, NGluRS retained the tRNAGln discriminatory property of EcGluRS. The discrimination was also shown to be present for a chimeric protein where NGluRS was attached to the anticodon-binding domain of EcGlnRS [14]. The fact that the catalytic domain retains discrimination in the absence of the cognate anticodon-binding domain as well as in the presence of the non-cognate anticodon-binding domain indicates that tRNA discriminatory elements are present in the catalytic domain of EcGluRS. Therefore, both the anticodon-binding domain and the catalytic domain contribute to tRNA discrimination. A comparison of crystal structures of GluRS in T. thermophilus (D-GluRS) and T. elongatus (ND-GluRS) did not reveal any significant difference in their catalytic domains, in terms of their differential tRNA specificity [12]. Rather, the anticodon-binding domains of the two proteins clearly showed how the presence of a conserved Arg residue in D-GluRS (Arg358 in TtGluRS) and its absence in ND-GluRS (Gly366) might lead to the discrimination of tRNAGln by D-GluRS. An Arg residue is also present in the anticodon-binding domain of EcGluRS at the corresponding position (Arg350 in EcGluRS). In addition, the presence of Ser (Ser438) in EcGluRS at a position corresponding to Gly417 in H. pylori GluRS2 also suggests that the anticodon-binding domain of EcGluRS is discriminatory against tRNAGln. Yet, without the anticodon-binding domain (NGluRS), EcGluRS still retained the capability to discriminate against tRNAGln. Our results are consistent with the work presented by Lee and Hendrickson [13] who showed that mutating a key Arg residue in the anticodon-binding domain of GluRS1 of H. pylori did not affect its tRNAGln discrimination. The catalytic domain of GluRS (NGluRS in EcGluRS) is considered to be the ancestral domain in GluRS, originally non-discriminatory, from which extant discriminating and non-discriminating GluRS evolved by anticodon-binding domain acquisition [3], [4]. Our result, that the catalytic domain of EcGluRS is inherently discriminatory against EctRNAGln, is significant and calls for a detailed bioinformatics study, focusing on the origin of this discrimination at residue level and the evolution of tRNAGln discrimination in bacteria.