MECHANISM OF TRP GENE REGULATION BY TRAP-RNA RECOGNITION

转录后调控机制在基因中发挥作用 可能在所有生物体中都有表达。枯草芽孢杆菌的陷阱调节 转录抑制和转录抑制表达trpEDCFBA操纵子 翻译控制机制。当被色氨酸激活时，TRAP结合到一个 5‘茎环(5’SL)和11(G/U)AG在新生的Trp先导中重复 转录，从而促进RNA聚合酶之前的转录终止 能达到色氨酸结构基因(衰减)。陷阱结合也促进了 形成一种抑制trpE翻译的RNA结构。这些机制 将进一步分析，以更好地了解枯草杆菌色氨酸蛋白 操纵子调控与转录后调控的多样性 机械装置。由于人类的几种疾病是由隔离的 三联体重复RNA结合蛋白结合的研究结果表明 HIV和几个癌基因的数量受到衰减的调节，结果来自这些 研究将间接有助于改善人类健康。 5‘SL在色氨酸操纵子表达中的作用将被研究。这个 TRAP中与5‘SL以及5’SL相互作用的氨基酸残基 与TRAP相互作用的核苷酸将由TRAP-5‘SL RNA结合决定 学习。5‘SL增加TRAP-Trp引导率的可能性 还将通过进行结合分析来检查RNA的相互作用。在……里面 此外，还将进行体外和体内实验，以确定 5‘SL作为RNA聚合酶暂停信号和/或mRNA不稳定信号 行列式。陷阱依赖所介导的翻译控制机制 形成trpE Shine-Delgarno(SD)阻挡发夹也将 检查过了。将进行体内表达研究，以确定是否 5‘SL参与了这一机制。体内表达，体外翻译， 还将进行信使核糖核酸半衰期实验，以确定 TrpE SD阻断发夹调控下游基因的表达 通过翻译偶联、转录极性和/或减少mRNA 稳定性。此外，还将进行实验，以确定是否 翻译控制需要更高的色氨酸浓度。 转录衰减所必需的。最后，RNA的可能作用 假结调控色氨酸操纵子的翻译和/或mRNA稳定性 将由几种遗传和生化方法决定。

Post-transcriptional regulatory mechanisms play a role in gene expression in probably all organisms. TRAP of Bacillus subtilis regulates expression of the trpEDCFBA operon by transcription attenuation and translational control mechanisms. When activated by tryptophan, TRAP binds to a 5' stem-loop (5'SL) and 11 (G/U)AG repeats in the nascent trp leader transcript, thereby promoting transcription termination before RNA polymerase can reach the trp structural genes (attenuation). TRAP binding also promotes formation of an RNA structure that inhibits trpE translation. These mechanisms will be further analyzed to gain a better understanding of B. subtilis trp operon regulation and the diversity of post-transcriptional regulatory mechanisms. Since several human disorders are caused by sequestration of triplet repeat RNA-binding proteins, combined with the findings that expression of HIV and several oncogenes is regulated by attenuation, results from these studies will indirectly contribute to improving human health. The role that the 5'SL plays in trp operon expression will be investigated. The amino acid residues of TRAP that interact with the 5'SL, as well as the 5'SL nucleotides that interact with TRAP will be determined by TRAP-5'SL RNA binding studies. The possibility that the 5'SL increases the rate of TRAP-trp leader RNA association will also be examined by performing binding assays. In addition, in vitro and in vivo experiments will be performed to determine if the 5'SL serves as an RNA polymerase pause signal and/or an mRNA instability determinant. The mechanism of translational control mediated by TRAP-dependent formation of the trpE Shine-Delgarno (SD) blocking hairpin will also be examined. In vivo expression studies will be carried out to determine if the 5'SL participates in this mechanism. In vivo expression, in vitro translation, and mRNA half-life experiments will also be performed to determine if formation of the trpE SD blocking hairpin regulates expression of the downstream genes via translational coupling, transcriptional polarity and/or decreasing mRNA stability. In addition, experiments will be carried out to determine if translational control requires a higher tryptophan concentration than is required for transcription attenuation. Finally, the possible role of an RNA pseudoknot in regulating translation and/or mRNA stability of the trp operon will be determined by several genetic and biochemical approaches.