application) The candidate for this 'Mentored Quantitative Research Career Development Award' is a physicist with a background that includes both academic and industrial research. His academic work as a graduate student (Cornell University), postdoc and Research Assistant Professor (University of Southern California) was in the field of low temperature physics. Using specialized nuclear magnetic resonance techniques he studied the thermal relaxation and excess magnetization properties of 3He surface layers. The industrial work (Raytheon Research Division) was in developing liquid crystal optics that can dynamically focus, steer and split laser beams under program control. His biology training has been obtained through course work (Washington University, Harvard University Extension School) and laboratory interactions with the proposed mentor, Jeff Gelles (Brandeis University, Biochemistry Department). The candidate intends to use the award period to further his goal of obtaining the background and credentials necessary for pursuing academic work in biophysics. Enhancer-mediated initiation is the most common means for regulating gene expression in eukaryotes. It is important to understand enhancer mechanisms, since gene regulation is central to processes ranging from tissue differentiation to the disease response. The proposed research examines enhancer-based transcription initiation in a simple bacterial system, where the initiation pathway may be studied in vitro using a minimal number of components. In enteric bacteria, nitrogen deprivation can initiate formation of an activator comprised of phosphorylated NtrC protein subunits. This activator assembles at an enhancer sequence and catalyzes the closed-to-open transition for the sigma54-RNA polymerase bound at the downstream promoter for the glnALG operon. The present research will use single molecule methods to elucidate the steps and intermediates involved with the initiation process. It will use single molecule fluorescence microscopy as a primary tool for recording and characterizing the NtrC activator assembly, and for observing the frequency and duration of the activator-polymerase binding events. It will also develop a single molecule assay for detecting the transcription initiated by an NtrC activator. The combined experiments will identify the oligomeric state of the functional activator and determine rate constants for the activator-polymerase bindings that lead to initiation. The single-molecule approach will highlight relations between the mechanisms at successive steps, thus contributing to an integrated description of the enhancer-mediated initiation process in this model system.
| Friedman, Larry J; Gelles, Jeff (2012) Mechanism of transcription initiation at an activator-dependent promoter defined by single-molecule observation. Cell 148:679-89 |
