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

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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.