Analysis of Regulatory Mechanism Controlling Tryptophan Metabolism in Bacteria

0093023查尔斯·亚诺夫斯基这项研究将集中于三个主要目标。第一个目标是鉴定枯草芽孢杆菌的YczA调节蛋白，并确定它是如何使TRAP调节蛋白失活的。TRAP蛋白调节6个色氨酸生物合成基因的转录，它在翻译上调节第7个色氨酸基因。YczA蛋白是由yczA-ycbK操纵子产生的，这是一种功能未知的操纵子。这个操纵子受转录衰减的调节；它在转录上被激活，以响应不带电的tRNATrp的积累。第二个目标是继续研究大肠杆菌和其他细菌物种用来调节降解性色氨酸酶(TNA)操纵子表达的转录衰减机制的详细特征。在大肠杆菌培养中添加色氨酸可以诱导TNA操纵子的表达。诱导导致抑制前导肽-tRNA的切割，这种抑制使翻译核糖体停滞。停滞的核糖体阻止Rho介导的转录终止在TNA操纵子的前导区。PI的实验室已经成功地在体外复制了这个操纵子的调节功能，现在计划详细检查肽基-tRNA裂解是如何被阻止的。第三个目标是使用枯草芽孢杆菌整个基因组的DNA微阵列分析来确定哪些基因因色氨酸代谢的变化而选择性地转录激活和转录抑制，就像PI最近对大肠杆菌基因组所做的那样。这些发现应该可以比较色氨酸代谢基因在两种微生物中的表达，这两种微生物是实验分析的首选对象。

0093023 Charles YanofskyThis research will focus on three principle objectives. The first objective is to characterize the YczA regulatory protein of Bacillus subtilis and determine how it inactivates the TRAP regulatory protein. The TRAP protein regulates transcription of six of the tryptophan biosynthetic genes, it regulates the seventh trp gene translationally. The YczA protein is produced by the yczA-ycbK operon, an operon of previously unknown function. This operon is regulated by transcription attenuation; it is transcriptionally activated in response to the accumulation of uncharged tRNATrp. The second objective is to continue studies on the detailed features of the mechanism of transcription attenuation used by E. coli and other bacterial species to regulate expression of the degradative tryptophanase(tna) operon. Tryptophan addition to E. coli cultures induces expression of the tna operon. Induction leads to inhibition of cleavage of a leader peptidyl-tRNA and this inhibition stalls the translating ribosome. The stalled ribosome prevents Rho mediated transcription termination in the leader region of the tna operon. The PI's laboratory has successfully reproduced the regulatory features for this operon in vitro and now plan to examine in detail exactly how peptidyl-tRNA cleavage is blocked. The third objective is to use DNA microarray analyses with the entire genome of Bacillus subtilis to determine which genes are selectively transcriptionally activated and transcriptionally inhibited in response to changes in tryptophan metabolism, as the PI recently did with the genome of E. coli. These findings should allow the comparison of expression of the genes of tryptophan metabolism in two microorganisms that are preferred subjects for experimental analysis.