The Interactions of TRAP with RNA to Regulate the trp Genes in Bacilli

这项研究的广泛，长期目标是了解蛋白质识别和结合特定RNA的机制，并研究这些相互作用如何调节基因表达。 研究的模型系统是TRAP蛋白（trp RNA结合衰减蛋白），一种调节芽孢杆菌中色氨酸生物合成基因表达的RNA结合蛋白。 TRAP通过以色氨酸依赖性方式结合到特定的RNA靶位点来负调节这些基因的转录和翻译。TRAP通过衰减机制控制trpEDCFBA操纵子的转录，该衰减机制涉及在5'前导区中形成的两个相互排斥的RNA二级结构：rho非依赖性转录终止子和重叠的抗终止子。 在存在过量色氨酸的情况下，TRAP结合前导转录物中的靶标，所述前导转录物由被2-3个非保守“间隔区”残基分隔的11个G/UAG重复组成。 这种结合促进终止子的形成，从而停止操纵子的转录。 TRAP还通过改变RNA二级结构或直接与核糖体竞争结合mRNA来调节几种trp基因的翻译。 TRAP含有11个相同的亚基，排列成对称的环，并通过结合相邻亚基之间口袋中的11个色氨酸分子而被激活以结合RNA。 TRAP与53个碱基RNA结合的晶体结构（分辨率为1.9埃），该RNA含有由Au间隔区隔开的11个GAG重复序列，该晶体结构已经回答了关于TRAP与RNA相互作用的许多问题，然而，关于这种不寻常的蛋白质/RNA相互作用的几个重要问题仍然没有回答。 在这个项目中，结晶学，生物化学和遗传学的方法相结合，将被用来继续分析这种新的RNA结合基因调控蛋白。这项工作将提供有关蛋白质如何识别RNA以及RNA结合蛋白如何控制基因表达的新信息。TRAP/RNA复合物的高分辨率晶体结构以及TRAP/RNA相互作用的热力学研究使其成为进行进一步详细分析的优秀系统。 目前，很少有其他的RNA结合蛋白，如果有任何调节基因表达，可以在此详细研究。 本项目的具体目标是：1）了解TRAP/RNA相互作用的特异性，包括G/UAG三联体第一个位置的G和U特异性的基础以及G/UAG重复之间的3个间隔核苷酸的作用。 2)确定疏水和堆积相互作用在TRAP/RNA复合物中的作用。 3)确定大分子水合作用在TRAP/RNA相互作用中的作用。蛋白质和RNA分子之间的特异性相互作用在调节所有生物体中的基因表达中起着至关重要的作用。 近年来，这些相互作用的重要性日益明显。 因此，RNA-蛋白质相互作用的研究对于理解控制基因表达的机制至关重要。 这项研究的长期目标是了解蛋白质识别和结合特定RNA的机制，并研究这些相互作用如何调节基因表达。 研究的模型系统是TRAP蛋白（trp RNA结合衰减蛋白），这是一种RNA结合蛋白，可调节称为芽孢杆菌的细菌中色氨酸生物合成基因的表达。TRAP是一种新的蛋白质，含有11个相同的亚基排列在一个环中，它识别并结合RNA，含有多个重复序列的GAG或UAG。 在这个项目中，结晶学，生物化学和遗传学的方法相结合，将被用来继续分析这种新的RNA结合基因调控蛋白。这项工作将提供有关蛋白质如何识别RNA以及RNA结合蛋白如何控制基因表达的新信息。TRAP/RNA复合物的高分辨率晶体结构，以及TRAP/RNA相互作用的热力学研究，使其成为对该系统进行进一步详细分析的优秀系统。目前，很少有其他的RNA结合蛋白，如果有任何调节基因表达，可以在此详细研究。

GollnickThe broad, long term goals of this research are to understand the mechanisms by which proteins recognize and bind to specific RNAs, and to investigate how these interactions regulate gene expression. The model system of study is the TRAP protein (trp RNA-binding Attenuation Protein), an RNA-binding protein that regulates expression of the tryptophan biosynthetic genes in Bacilli. TRAP negatively regulates both transcription and translation of these genes by binding to specific RNA target sites in a tryptophan dependent manner. TRAP controls transcription of the trpEDCFBA operon via an attenuation mechanism involving two mutually exclusive RNA secondary structures that form in the 5' leader region: a rho-independent transcription terminator and an overlapping antiterminator. In the presence of excess tryptophan, TRAP binds to a target in the leader transcript consisting of 11 G/UAG repeats separated by 2-3 nonconserved "spacer" residues. This binding promotes formation of the terminator, halting transcription of the operon. TRAP also regulates translation of several trp genes by either altering the RNA secondary structure or by directly competing with ribosomes for binding to the mRNA. TRAP contains 11 identical subunits arranged in a symmetrical ring and is activated to bind RNA by binding 11 tryptophan molecules in pockets between adjacent subunits. The crystal structure (to 1.9 angstrom resolution) of TRAP bound to a 53 base RNA containing 11 GAG repeats separated by AU spacers has answered many questions about the interaction TRAP with RNA, however several important questions about this unusual protein/RNA interaction remain unanswered. In this project, a combination of crystallography, biochemical and genetic approaches will be used to continue analysis of this novel RNA-binding gene regulatory protein. This work will provide new information about how proteins recognize RNA and how RNA-binding proteins control gene expression. The high resolution crystal structure of a TRAP/RNA complex, together with thermodynamic studies of the TRAP/RNA interaction makes it an excellent system with which to perform further detailed analyses. Currently, there are few, if any other RNA-binding proteins that regulate gene expression that can be studied in this detail. The specific aims of the project are: 1) Understand the specificity of the TRAP/RNA interaction including the basis for the specificity for G and U in the first position of the G/UAG triplets and the effect of 3 spacer nucleotides between the G/UAG repeats. 2) Determine the role of hydrophobic and stacking interactions in the TRAP/RNA complex. 3) Determine the role of macromolecular hydration in the TRAP/RNA interaction. Specific interactions between proteins and RNA molecules play crucial roles in regulating gene expression in all organisms. The importance of these interactions has become increasingly clear in recent years. The study of RNA-protein interactions is therefore crucial to understand the mechanisms that control gene expression. The broad, long term goals of this research are to understand the mechanisms by which proteins recognize and bind to specific RNAs, and to investigate how these interactions regulate gene expression. The model system of study is the TRAP protein (trp RNA-binding Attenuation Protein), an RNA-binding protein that regulates expression of the tryptophan biosynthetic genes in bacteria called Bacilli. TRAP is a novel protein containing 11 identical subunits arranged in a ring and it recognizes and binds to RNAs that contain multiple repeats of the sequence GAG or UAG. In this project, a combination of crystallography, biochemical and genetic approaches will be used to continue analysis of this novel RNA-binding gene regulatory protein. This work will provide new information about how proteins recognize RNA and how RNA-binding proteins control gene expression. The high resolution crystal structure of a TRAP/RNA complex, together with thermodynamic studies of the TRAP/RNA interaction makes it an excellent system with which to perform further detailed analyses of this system. Currently, there are few, if any other RNA-binding proteins that regulate gene expression that can be studied in this detail.