RNA synthesis, or transcription, is the main target for regulation of gene expression in all three kingdoms of life. The RNA polymerases of bacteria, archaea and eukaryotes, which are responsible for RNA synthesis, are strikingly similar in structure. The aim of this research program is to gain a detailed understanding of how the RNA polymerase from the bacterium, Escherichia coli, forms a transcription-competent complex at promoter DNA, the site of initiation of RNA synthesis. Such information is vital to our detailed understanding of RNA polymerase's mechanism of action and in turn, how gene expression is regulated. In view of the similarities of their RNA polymerases, this information will be relevant to all bacteria, including pathogens.
Broader Impact: This research program affords the opportunity to maintain a strong representation of women and underrepresented minorities. To extend this to undergraduate students as well, students are recruited through the Summer Program in Undergraduate Research (SPUR) at Case Western Reserve University. This program makes it possible for students to spend ten weeks working in a research laboratory under the direction of a faculty member, culminating in the presentation of a poster in the final week. The students are provided a stipend to defray the costs of their participation.
The funded proposal was named "Interaction of E. coli RNA polymerase with promoter DNA to form an initiation-competent complex". Our original goal was to work on several projects having to do with how the enzyme RNA Polymerase gets ready to make RNA, an important step in going from gene to protein. However, here I will not discuss the original project. At my request, we got permission to also study biofilms, which are formed when a bacterium (Escherichia coli in our case) forms dense cultures on surfaces. It is important to study biofilms, as they are a clinical problem. For example the biofilm may coat the inside of indwelling (i.e. in the body) catheters, and thus block passage urine or also of important medicinal fluids to a patient. Others had determined that the Ag43 protein plays an important role in biofilm formation. In order to make the Ag43 protein, Ag43 messenger RNA has to be synthesized. However in this case the DNA of the E. coli specifies that between the start site of RNA synthesis and the agn43 gene there is a "terminator", which would halt further RNA synthesis and prevent Ag43 protein from being made. We hypothesized that the bacterium must be able to regulate this terminator so that under conditions when the bacterium is making a biofilm there is good production of mRNA and of the Ag43 protein. However at other times, when the Ag43 protein is not needed, the terminator would be active and the agn43 messenger RNA would not be made. The results we obtained supported our hypothesis. Under conditions conducive for biofilm formation, we found that after RNA synthesis was initiated, it would progress past the terminator. Thus, under these conditions, the cell must have a way to allow RNA polymerase to bypass the terminator. On the other hand, the opposite was found to be true under conditions that would not prompt the E. coli bacterium to make biofilms: RNA synthesis did not extend past the terminator and Agn43 protein would not be made. We do not yet know how the cell is able to allow terminator bypass by the RNA polymerase. In bacteriophages (viruses that infect bacteria) two ways have been found to bypass terminators of RNA synthesis. One is that the structure of the RNA adopts a shape that makes the terminator inactive. Another is that in the cell a protein is made that attaches to the RNA polymerase and now enables the RNA polymerase to bypass the terminator. We think it is very likely that one of these two ways are at work here as well. Our preliminary data suggest that the latter possibility may be the case here. Broader Impact During the 4 years of the grant, 4 women have worked on various projects: Disha Haque, undergraduate (now at The Ohio State University, and soon to be in a medical school; no choice made yet) Manasa Sagaram, undergraduate (Now doing a post-bac at the University of Toledo) Heather Oreh, BA (now taking care of a special needs child). Tyrishea Crumedy (under represented minority) high school student (now at Central State University), and 1 man: Ebenezer Minaya (under represented minority) For the two undergraduates it was the their first time doing research. The two undergraduate students had some experience. Howwever, when each of them had just arrived arrived in the laboratory , the advice of my technician Heather Oreh's and myself was essential. All got useful results, but they did not accumulate enough data to be included among the authors of the paper we published. The two high school students also benefitted from lectures presented in the program "Scientific Enrichment and Opportunity Program designed to bring underprivileged students from the Cleveland Metropolitan School District into contact with faculty at the School of Medicine".
