MECHANISM OF TRP GENE REGULATION BY TRAP-RNA RECOGNITION

TRAP-RNA识别调控TRP基因的机制

• 批准号: 6193058
• 负责人: PAUL L BABITZKE
• 金额: $ 19.98万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6193058
• 关键词: Bacillus subtilis; RNA binding protein; SDS polyacrylamide gel electrophoresis; bacterial RNA; bacterial genetics; bacterial proteins; double stranded RNA; gel mobility shift assay; gene mutation; genetic regulation; genetic translation; nucleic acid repetitive sequence; nucleotides; operon; protein structure function; ribosomes; transcription termination

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.

转录后调控机制在基因表达调控中发挥作用 可能在所有生物体中都有表达。枯草芽孢杆菌的TRAP调节 通过转录衰减表达trpEDCFBA操纵子， 翻译控制机制当被色氨酸激活时，TRAP结合到 在新生的trp前导序列中存在5'茎环（5' SL）和11（G/U）AG重复序列 转录物，从而在RNA聚合酶之前促进转录终止 可以到达Trp结构基因（衰减）。TRAP结合还促进 形成抑制trpE翻译的RNA结构。这些机制 将进一步分析，以更好地了解B。枯草杆菌色氨酸 操纵子调控和转录后调控的多样性 机制等由于几种人类疾病是由螯合 三联重复RNA结合蛋白，结合表达的发现， 艾滋病毒和几个癌基因的调节衰减，结果从这些 研究将间接有助于改善人类健康。 将研究5 'SL在trp操纵子表达中所起的作用。的 与5 'SL相互作用的TRAP的氨基酸残基，以及5' SL 与TRAP相互作用的核苷酸将通过TRAP-5’SL RNA结合来确定 问题研究5 'SL增加TRAP-trp前导序列速率的可能性 还将通过进行结合测定来检查RNA缔合。在 此外，将进行体外和体内实验以确定是否 5’SL作为RNA聚合酶暂停信号和/或mRNA不稳定性 行列式TRAP依赖性蛋白介导的翻译调控机制 trpE Shine-Delgarno（SD）阻断发夹的形成也将是 考察将进行体内表达研究以确定所述表达产物是否为真核表达。 5 'SL参与该机制。体内表达，体外翻译， 还将进行mRNA半衰期实验以确定是否形成 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/或降低mRNA 稳定此外，将进行实验，以确定是否 翻译控制需要比 转录衰减所需的。最后，RNA的可能作用 调节色氨酸操纵子翻译和/或mRNA稳定性的假结 将通过几种遗传和生物化学方法来确定。