Bacteria rely on numerous global gene regulators to provide rapid benefit or protection from sudden environmental change. DksA, an atypical bacterial gene regulator, plays an essential role in the regulation of transcription (RNA synthesis) of ribosomal RNA (necessary for the synthesis of proteins in the cell) and of other genes involved in important cellular processes. DksA differs from most bacterial gene regulators in that it binds directly to RNA polymerase (the enzyme responsible for carrying out transcription) rather than to DNA. Little is known about the regulation of DksA itself. Therefore, this project focuses on attaining a detailed understanding of how Escherichia coli cells control the production of DksA in response to different growth conditions. Preliminary work indicated that the dksA gene is regulated at the level of transcription. The regulation involves two transcription initiation sites, and several other transcription factors. The dksA gene expression was also found to be controlled at the level of translation (protein synthesis), and this control required a specific portion of the dksA mRNA referred to as the 5'-untranslated region (5'-UTR). One aim of this project is to investigate how dksA is transcriptionally controlled under various different growth conditions. The dksA promoters, which control the initiation of transcription, including a newly discovered promoter, which is most active under conditions of starvation, will be more precisely defined. The dksA regulatory effects by several suspected transcription factors will be investigated in detail. The relative transcription activity from all dksA promoters will be simultaneously measured while in their native chromosomal contexts during different growth conditions and in different bacterial strains lacking any of suspected transcriptional regulators. Another aim is to investigate how dksA gene expression is controlled after the dksA transcript (mRNA) has been made. The project will examine the hypothesis that changes in the rate of breakdown of dksA mRNA during different phases of cell growth contribute to the regulation of dksA expression. The relative DksA protein levels in various E. coli strains and several other bacterial species will be measured during different growth conditions. The effects of suspected dksA transcription regulators on the cellular levels of DksA protein will also be examined. The RNA structure in the 5'-UTR of the dksA mRNA will be studied. The project also aims to detect, capture, and identify potential cellular factors that may associate with the 5'-UTR to affect translation control of DksA. Together, these experiments will provide a broad and extensive understanding of the cellular processes used to regulate this important global gene regulator in response to different bacterial growth conditions.
Broader Significance: Knowledge gained from this work will be applicable to other bacteria that are of environmental, industrial, or agricultural importance. Broader contributions will include research training of four graduate and eight to twelve undergraduate students, as part of their education experience at SUNY, Albany. Research training provided by the PI has thus far resulted in five doctoral graduates, three masters graduates, and twenty three research-trained undergraduate students of diverse race, ethnicity, and gender, that have subsequently succeeded in securing research, academic, or professional careers, or have continued on to acquire graduate or professional education. Synergy between research and teaching will also occur in the role of the PI as instructor of an undergraduate biochemistry laboratory course. The PI will train students in the application of molecular and biochemical approaches, some of which will be used to address aspects of this project. As a member of an underrepresented group, the PI is in a distinctive position to serve as a role model and mentor for underrepresented students wishing to pursue a career in the sciences. In his roles as coordinator of the undergraduate research program, coordinator of the biochemistry and molecular biology major, and instructor for a biochemistry lab course, the PI interacts with a number of minority undergraduate students with strong academic skills and motivation for research, among which several students will be enlisted to participate in this project and receive research training. The PI will interact with university campuses in Puerto Rico to recruit students to our graduate program and to this research project. Thus, underrepresented graduate and undergraduate students will be trained to conduct research in this project. Research training supports our nation's research infrastructure and increases appreciation and awareness of the importance of basic science research.
