Mechanism of trp Gene Regulation by TRAP-RNA Recognition

描述(申请人提供)：转录后调控机制在可能所有生物体的基因表达中发挥作用。枯草芽孢杆菌的TRAP通过转录抑制和翻译调控机制调节色氨酸的生物合成和运输。当被色氨酸激活时，TRAP与新生trpEDCFBA操纵子前导转录本中的5‘茎环(5’SL)和11个(G/U)AG重复序列结合，从而在RNA聚合酶到达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)暂停参与了衰减和trpE翻译控制机制。此外，在PABA(TrpG)、trpP(YhaG)和ycbK转录本中，TRAP与多个三联体重复序列的结合通过直接阻止核糖体结合来调节翻译。这些机制将被进一步分析，以获得更全面的了解枯草杆菌是如何调节色氨酸代谢的，以加强我们对转录后调控机制的多样性的了解，并确定决定蛋白质-RNA识别的一些基本原理。由于一些人类疾病是由三重重复RNA结合蛋白的隔离引起的，再加上艾滋病毒和几个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学和生化方法相结合的方法进行研究。将通过测定TRAP与5‘SL相互作用的氨基酸残基以及与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，还将进行实验以确定5‘SL是否作为mRNA不稳定的决定因素。还将研究TRAP介导的ycbK翻译控制的机制。虽然PABA、trpP和ycbK依赖于陷阱的翻译控制机制相似，但很明显，三联体重复的相对排列导致了不同程度的陷阱介导的控制。将进行遗传和生化实验，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.