RUI: Evolution of Bacterial Asparaginyl-tRNA Synthesis

这项研究的目的是了解为什么某些细菌使用两种不同的方法来制备蛋白质合成所需的氨基酸天冬酰胺。这些结果将为深入了解这些替代途径的进化起源，以及它们如何赋予在不同自然环境中生长的细菌适应生理优势，即在土壤中与哺乳动物宿主内生长。包括少数族裔在内的本科生将通过该项目接受实验室研究方面的培训。培训的影响将通过在科学会议上发表研究报告、学生与他人共同撰写同行评议的文章以及未来学生在劳动力和研究生项目中的安置来衡量。此外，该项目将允许将研究中产生的研究整合到实验生物化学实验室课程中，培训更多的本科生进行假设驱动的生物化学研究。为了扩大科学素养，并在STEM学科中留住更多来自代表性不足的少数民族的学生，该项目将向中学生提供外联服务。将遗传信息翻译成蛋白质的氨基酸序列对细胞生命至关重要。这一过程的保真度取决于正确的适配器分子--氨基酰-tRNAs的形成。在细胞中，主要通过氨酰-tRNA合成酶将氨基酸连接到正确的tRNA上。每个tRNA合成酶只针对一种氨基酸，只将该氨基酸连接到一组特定的tRNA分子上。然而，在许多细菌基因组中，直接将天冬酰胺结合到其同源tRNA上的天冬酰胺-tRNA合成酶并没有编码。相反，这些生物通过间接的两步途径在tRNA上合成天冬酰胺。首先，他们使用一种无区别的天门冬氨酰-tRNA合成酶来将tRNA与天冬氨酸进行氨基酰化反应。然后，与tRNA结合的天冬氨酸被酰胺转移酶GatCAB酰胺化，形成天冬酰胺基tRNA。许多细菌，包括枯草芽孢杆菌和嗜盐芽孢杆菌，编码了天冬酰胺-tRNA合成的两条途径。编码这两条路线的细菌的一个子集获得了古生菌的非区分天冬氨酰-tRNA合成酶，用于依赖tRNA的天冬酰胺生物合成。该项目的目标是使用生化和微生物遗传学方法来阐明为什么如此多的细菌保留了天冬酰胺-tRNA形成的两条途径，以及为什么某些细菌获得了古生菌的非区分天冬氨酰-tRNA合成酶来完成这一任务。预计结果将有助于阐明对蛋白质合成的准确性至关重要的过程的演变。

The goal of this research is to understand why certain bacteria employ two distinct routes for preparing the amino acid asparagine for protein synthesis. The results will provide insights into the evolutionary origin of these alternate pathways and how they may confer adaptive physiological advantages to bacteria growing in different natural environments, i.e., in soil versus inside a mammalian host. Undergraduate students, including members of underrepresented minorities, will be trained in laboratory research through the project. The impact of the training will be measured by the presentation of the research at scientific meetings, student co-authorship of peer reviewed articles, and future student placement in the workforce and in graduate programs. In addition, the project will allow studies arising from the research to be integrated into an experimental biochemistry laboratory course, training additional undergraduate students in hypothesis driven biochemical research. To expand scientific literacy and retain more students from underrepresented minorities in STEM disciplines, the project will provide outreach to middle school students. Translation of a genetic message into the amino acid sequence of a protein is essential for cellular life. The fidelity of the process is dependent on the formation of the correct adaptor molecules, aminoacyl-tRNAs. Attaching an amino acid to the right tRNA is carried out in cells primarily by aminoacyl-tRNA synthetases. Each tRNA synthetase is specific for one amino acid and only ligates the amino acid onto a certain set of tRNA molecules. However, in many bacterial genomes asparaginyl-tRNA synthetase that directly attaches asparagine to its cognate tRNA is not encoded. Instead these organisms synthesize asparagine on the tRNA via an indirect two-step pathway. First they use a non-discriminating aspartyl-tRNA synthetase to aminoacylate tRNA with aspartate. The tRNA-bound Asp is then amidated by the amidotransferase GatCAB to form asparaginyl-tRNA. A number of bacteria, including Bacillus subtilis and Bacillus halodurans, encode both routes for asparaginyl-tRNA synthesis. A subset of bacteria encoding both routes acquired an archaeal non-discriminating aspartyl-tRNA synthetase for tRNA-dependent asparagine biosynthesis. The objectives of this project are to use biochemical and microbial genetic approaches to elucidate why so many bacteria retain both routes for asparaginyl-tRNA formation and why certain bacteria acquired an archaeal non-discriminating aspartyl-tRNA synthetase for the task. Results are expected to shed light on the evolution of a process that is crucial for the accuracy of protein synthesis.