Mechanism of trp Gene Regulation by TRAP-RNA Recognition

描述（由申请人提供）：转录后调控机制可能在所有生物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trpEDCFBA操纵子先导转录体中的5'茎环（5sl）和11 (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结合也促进抑制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，TRAP结合到pabA （trpG）、trpP （yhaG）和ycbK转录本中的多个三联体重复序列上，通过直接阻断核糖体结合来调节翻译。我们将进一步分析这些机制，以更全面地了解枯草芽孢杆菌如何调节色氨酸代谢，加强我们对转录后控制机制多样性的认识，并确定一些决定蛋白质- rna识别的基本原理。由于一些人类疾病是由三联体重复rna结合蛋白的隔离引起的，再加上艾滋病毒和几种致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。

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.