Substrate Divergence in Aminoacyl-tRNA Biosynthesis

描述(由申请人提供)： 将遗传密码准确地翻译成功能多肽依赖于分子识别中的关键事件。氨基酰-tRNA合成酶(AARs)是这一过程的核心。这些酶催化特定的tRNA氨酰化反应。他们的产物，氨酰-tRNAs，将核酸遗传密码转换为核糖体内的蛋白质。除了少数几个例外，每个AARS都对单一同源氨基酸和单一类别的tRNA同源受体具有精致的专一性。谷氨酰-tRNA合成酶(GluRS)是这一特异性规则的一个不寻常的例外；一些GluRS氨基酰化tRNA(Glu)，同源底物，和“非同源”tRNA(Gln)；在这些情况下，GluRS被称为非区分(GluRS-ND)。幽门螺杆菌的致病菌依赖于两个GluRS-D(GluRS1)和第二个GluRS(GluRS2)，GluRS既不区分也不区分。这种HP GluRS2错酰化tRNA(GIN)与谷氨酸，但不容易氨基酰化“同源”tRNA(Glu)。有人认为，这两个GluRS代表了现代GluRS-D进化的中间产物，以及未来可能的细菌谷氨酰tRNA合成酶(GInRS)。因此，GIuRS1和GluRS2可用于实验探索底物特异性的进化。本提案中描述的研究将定义导致这两种酶tRNA特异性微调的分子差异，同时描述GluRS-NDS和GluRS-DS之间的差异。此外，实验将直接探索GluRS2的氨基酸专一性，以确定这种酶如何选择谷氨酸而不是谷氨酰胺，询问它是否可以转化为具有功能的细菌GInRS。总之，这一提议将为密切相关的AARs的底物特异性之间的细微差异提供详细的分子洞察，同时提供对这些酶的进化可能如何进行的直接洞察。

DESCRIPTION (provided by applicant): Accurate translation of the genetic code into functional polypeptides relies on critical events in molecular recognition. The aminoacyl-tRNA synthetases (AARSs) are at the heart of this process. These enzymes catalyze specific tRNA aminoacylation reactions. Their products, the aminoacyl-tRNAs, convert the nucleic acid genetic code into proteins within the ribosome. With only a few exceptions, each of the AARSs is exquisitely specific for a single cognate amino acid and a single class of tRNA isoacceptors. Glutamyl-tRNA synthetase (GluRS) is an unusual exception to this specificity rule; some GluRSs aminoacylate both tRNA(Glu), the cognate substrate, and the "non-cognate" tRNA(Gln); in these cases, GluRS is termed non-discriminating (GluRS-ND). The pathogenic bacterium Helicobacter pylori relies on two GluRSs - a discriminating GluRS-D (GluRS1) and a second GluRS (GluRS2) that is neither discriminating nor non-discriminating. This Hp GluRS2 misacylates tRNA(GIn) with glutamate but does not readily aminoacylate "cognate" tRNA(Glu). It is proposed that these two GluRSs represent intermediates in the evolution of a modern GluRS-D and a possible future, bacterial glutaminyl-tRNA synthetase (GInRS). Thus, GIuRS1 and GluRS2 can be used to experimentally probe the evolution of substrate specificity. The research described in this proposal will define the molecular differences that result in the fine-tuned tRNA specificities of these two enzymes, while concomitantly delineating the differences between GluRS-NDs and GluRS-Ds. Additionally, experiments will directly probe the amino acid specificity of GluRS2 to determine how this enzyme selects glutamate over glutamine, asking whether or not it can be converted into a functional, bacterial GInRS. In total, this proposal will provide detailed molecular insight into the subtle differences between the substrate specificities of closely related AARSs, while providing direct insight into how the evolution of these enzymes might have proceeded.