Analysis of Regulatory Mechanism Controlling Tryptophan Metabolism in Bacteria

对全球基因表达的精细控制使细菌能够保持平衡的新陈代谢，并迅速适应环境挑战和不断变化的营养条件。对参与色氨酸生物合成或利用的基因的控制已成为发现和表征细菌新的遗传调控机制的最重要的范例之一。不同的机制既可以检测游离色氨酸的可用性，也可以检测tRNATrp的充电水平(氨基酰化)。该项目将通过研究大肠杆菌中TNA操纵子(色氨酸的利用)和地衣芽孢杆菌中的AT操纵子(色氨酸调节)来继续这些研究。将使用TNA操纵子进行结构和调控研究，以确定新生的TNAC肽-tRNA和核糖体出口隧道中的分子特征，这些分子特征负责色氨酸与核糖体的肽转移酶中心结合，以及伴随而来的TNAC终止密码子对肽-tRNA切割的抑制。将在rRNA和r-蛋白质中引入特定的核苷酸替换或氨基酸替换，以确定识别新生多肽和游离色氨酸特性所必需的核糖体决定因素。将使用一种高效的体外系统来检测纯化的核糖体。这个项目揭示了翻译核糖体不是一个非特异性的蛋白质合成机器。相反，多肽的氨基酸序列会影响核糖体催化多肽延长或终止密码子的切割的能力。还将对革兰氏阳性细菌地衣芽孢杆菌进行研究，以确定at操纵子前导肽编码区的调控特征。该编码区包含三个分散的色氨酸密码子，如果没有色氨酸tRNATrp，翻译核糖体可能会停滞。前两个色氨酸密码子的核糖体停滞预计会增加AT调节蛋白的合成，而第三个色氨酸密码子的核糖体停滞预计会减少AT的合成。该项目为博士后学生提供培训，并正在为微生物中基因表达的转录后调控建立新的范例。

The fine control of global gene expression allows bacteria to maintain a balanced metabolism and to adjust rapidly to environmental challenges and changing nutrient conditions. The control of the genes involved in tryptophan biosynthesis or utilization has served as one of the most important paradigms for the discovery and atomic level characterization of novel genetic regulatory mechanisms in bacteria. The diverse mechanisms sense both the availability of free tryptophan and the level of charging (aminoacylation) of tRNATrp. This project will continue these investigations by investigating the control of the tna operon (tryptophan utilization) in Escherichia coli and the at operon (tryptophan regulation) in Bacillus licheniformis. Structural and regulatory studies will be performed using the tna operon to identify the molecular features in the nascent TnaC peptidyl-tRNA and in the ribosome exit tunnel that are responsible for tryptophan binding to the peptidyl transferase center of the ribosome and the concomitant inhibition of peptidyl-tRNA cleavage at the tnaC stop codon. Specific nucleotide substitutions or amino acid replacements will be introduced in rRNA and r-proteins to identify the ribosomal determinants essential for recognition of the features of the nascent peptide and free tryptophan. An efficient in vitro system will be used in which purified ribosomes are examined. This project is revealing that the translating ribosome is not a non-specific protein-synthesizing machine. Rather, the amino acid sequence of a polypeptide can influence a ribosome's ability to catalyze polypeptide elongation, or cleavage at a stop codon. Studies will also be performed with the gram-positive bacterium Bacillus licheniformis to determine the regulatory features of the leader peptide coding region of the at operon. This coding region contains three dispersed tryptophan codons at which a translating ribosome could stall if tryptophan-charged tRNATrp is unavailable. Ribosome stalling at either of the first two tryptophan codons is predicted to increase synthesis of the AT regulatory protein whereas ribosome stalling at the third tryptophan codon is predicted to reduce AT synthesis. This project provides training for post doctoral students and is establishing new paradigms for the post transcriptional regulation of gene expression in microorganisms.