Regulation of the Bacillus Subtilis TRP Genes by an RNA Binding Protein

智力优势：在所有细胞中表达基因的第一步是将储存在DNA基因组中的信息复制到RNA的中间分子中。这一过程被称为转录，在所有生物体中都受到高度调控。转录从DNA上称为启动子的位置开始，在终止子结束。虽然人们对启动过程以及如何对其进行监管知之甚多，但对终止过程的了解相对较少。在许多细菌中，基因的表达受到一个称为衰减的过程的调节，该过程在基因的蛋白质编码部分转录之前提前终止转录。这个项目将研究一种这样的衰减机制，它控制一种名为枯草芽孢杆菌的细菌中参与合成氨基酸色氨酸(Trp)的基因的表达。这些基因的衰减控制是由一种名为TRAP的蛋白质调节的。当细菌细胞有足够的色氨酸时，TRAP与Trp RNA上的特定位置结合，导致转录在基因的蛋白质编码区之前终止(S)。以前的工作表明，TRAP发挥作用的机制是改变RNA的结构。然而，最近的研究表明，仅仅改变RNA结构并不足以导致这些基因的转录终止。因此，陷阱在导致终止过程中起着额外的作用。该项目的目标之一是确定TRAP在控制转录方面所起的作用。彻底阐明TRAP如何影响RNA聚合酶(转录酶)的终止，将对我们理解细菌转录的基本过程做出重要而独特的贡献。该项目的第二个目标是研究另一种名为反TRAP的蛋白质对TRAP蛋白的调节。抗TRAP通过与TRAP结合并阻止其与RNA结合来抑制TRAP的衰减活性。TRAP和抗TRAP均为多聚体蛋白，由相同的亚基组成(11个为TRAP，3个为抗TRAP)。最近的研究表明，多个反陷阱三聚体可以与陷阱11-聚体结合。然而，这一点的意义尚不清楚。这个项目将评估有多少反TRAP三聚体必须与TRAP结合，以防止它与RNA结合，以及这些蛋白质-蛋白质相互作用的扰动如何影响调控。更广泛的影响：研究生和本科生，包括妇女和代表不足的少数族裔学生将参加这些研究。布法罗大学是一个非常多元化的校园，为指导少数族裔学生提供了机会。这个项目的研究将由四名女学生进行，其中一名是非洲裔美国人，一名是西班牙裔。旅行资金将用于使学生能够在全国会议上发表演讲和张贴海报。这项工作的发现将包括在我们本科生遗传学课程的课堂上，以及我们研究生的高级分子遗传学课程中。这个项目之前的工作已经进入了本科生和研究生课程使用的几本教科书。我们完全有理由相信，这个项目的结果也会是一样的。

Intellectual Merit: The first step to express genes in all cells is to copy the information stored in the DNA genome into an intermediate molecule of RNA. This process is called transcription and is highly regulated in all organisms. Transcription starts at a location on the DNA called a promoter and finishes at a terminator. While a great deal is known about the process of initiation and how it is regulated, relatively less is known about the process of termination. In many bacteria, expression of genes is modulated by a process called attenuation, which prematurely terminates transcription prior to transcription of the protein coding portion of the gene. This project will examine one such attenuation mechanism that controls expression of the genes involved in the synthesis of the amino acid tryptophan (trp) in a bacterium called Bacillus subtilis. Attenuation control of these genes is modulated by a protein called TRAP. When the bacterial cell has sufficient tryptophan, TRAP binds to a specific site on the trp RNA and causes transcription to terminate prior to the protein coding region of the gene(s). Previous work suggested that the mechanism by which TRAP functions is to alter the structure of the RNA. However, recent studies have shown that just altering the RNA structure is not sufficient to cause transcription to terminate in these genes. Hence, TRAP plays an additional role in causing termination. One of the goals of this project is to define the role that TRAP plays in controlling transcription. Complete elucidation of how TRAP influences RNA polymerase (the transcription enzyme) to terminate will make an important and unique contribution to our understanding of the fundamental process of transcription in bacteria. A second goal of the project is to study regulation of the TRAP protein by another protein called Anti-TRAP. Anti-TRAP inhibits the attenuation activity of TRAP by binding to TRAP and preventing it from binding to RNA. Both TRAP and Anti-TRAP are multimeric proteins, composed of identical subunits (11 for TRAP and 3 for Anti-TRAP). Recent studies have shown that multiple Anti-TRAP trimers can bind to the TRAP 11-mer. However the significance of this is not clear. This project will evaluate how many Anti-TRAP trimers must bind to TRAP to prevent it from binding RNA and how perturbations of these protein-protein interactions affect regulation. Broader impacts: Graduate students and undergraduates, including women and underrepresented minority students will participate in these studies. The University at Buffalo is a very diverse campus, providing the opportunity to mentor minority students. The research for this project will be conducted by four women students, one of whom is African American and one who is Hispanic. Travel funds will be used to enable students to present talks and posters at national meetings. Findings from this work will be included in lectures in our undergraduate Genetics course, as well as in our graduate Advanced Molecular Genetics course. Prior work on this project has already made its way into several text books used in undergraduate and graduate courses. There is every reason to believe that the results from this project will do the same.