Mechanism of trp Gene Regulation by TRAP-RNA Recognition

描述（由申请人提供）： 转录后调节机制可能在所有生物体中的基因表达中发挥作用。枯草芽孢杆菌的TRAP通过转录衰减和翻译控制机制调节色氨酸的生物合成和转运。当被色氨酸激活时，TRAP与新生trpEDCFBA操纵子前导转录物中的5'茎环（5' SL）和11（G/U）AG重复序列结合，从而在RNA聚合酶可以到达trp操纵子结构基因之前促进转录终止（衰减）。TRAP结合还促进抑制trpE翻译的RNA结构的形成。NusA刺激的RNA聚合酶（RNAP）暂停参与衰减和trpE翻译控制机制。此外，TRAP与pabA（trpG）、trpP（yhaG）和ycbK转录物中的多个三联体重复的结合通过直接阻断核糖体结合来调节翻译。这些机制将进一步分析，以获得一个更完整的理解如何B。枯草杆菌调节色氨酸代谢，加强我们的知识的多样性转录后控制机制，并确定一些基本原则，决定蛋白质-RNA识别。由于几种人类疾病是由三重重复RNA结合蛋白的螯合引起的，结合HIV和几种癌基因的表达受衰减调节的发现，这些研究的结果将间接有助于改善人类健康。 RNAP暂停的机制及其在衰减和trpE翻译控制中的作用将使用遗传和生物化学方法的组合进行检查。将通过确定与5 'SL相互作用的TRAP的氨基酸残基和与TRAP相互作用的5' SL核苷酸来研究TRAP-5 'SL相互作用的机制。此外，将进行实验以确定5 'SL是否作为mRNA不稳定性决定簇。TRAP介导的ycbK翻译控制的机制也将被检查。虽然pabA、trpP和ycbK的TRAP依赖性翻译控制机制是相似的，但显然三联体重复序列的相对排列导致不同程度的TRAP介导的控制。将进行遗传和生物化学实验，以了解三联体重复序列的相对位置如何影响TRAP介导的这三个基因的翻译控制。

DESCRIPTION (provided by applicant): Post-transcriptional regulatory mechanisms play a role in gene expression in probably all organisms. TRAP of Bacillus subtilis regulates tryptophan biosynthesis and transport 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 trpEDCFBA operon leader transcript, thereby promoting transcription termination before RNA polymerase can reach the trp operon structural genes (attenuation). TRAP binding also promotes formation of an RNA structure that inhibits trpE translation. NusA-stimulated RNA polymerase (RNAP) pausing participates in the attenuation and trpE translation control mechanisms. In addition, TRAP binding to multiple triplet repeats in the pabA (trpG), trpP (yhaG) and ycbK transcripts regulates translation by directly blocking ribosome binding. These mechanisms will be further analyzed to gain a more complete understanding of how B. subtilis regulates tryptophan metabolism, to strengthen our knowledge about the diversity of post-transcriptional control mechanisms, and to determine some of the fundamental principles that dictate protein-RNA recognition. 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 mechanism of RNAP pausing and its role in attenuation and trpE translation control will be examined using a combination of genetic and biochemical approaches. The mechanism of TRAP-5'SL interaction will be investigated by determining the amino acid residues of TRAP that interact with the 5'SL and the 5'SL nucleotides that interact with TRAP. In addition, experiments will be performed to determine if the 5'SL serves as an mRNA instability determinant. The mechanism of TRAP-mediated ycbK translation control will also be examined. While the TRAP-dependent translation control mechanisms for pabA, trpP and ycbK are similar, it is apparent that the relative arrangement of the triplet repeats results in different degrees of TRAP-mediated control. Genetic and biochemical experiments will be carried out to understand how the relative placement of the triplet repeats influences TRAP-mediated translation control of these three genes.