Transcription: Mechanism and Regulation

PROJECT SUMMARY/ABSTRACT The work proposed is designed to understand how gene expression control at the transcriptional level results from alterations in the activity of RNA polymerase (RNAP). Escherichia coli RNAP serves as an exceptional model system for mechanistic studies of transcription and as a paradigm for understanding gene expression in bacteria. Bacteria live in complex, fluctuating environments requiring gene expression to be highly responsive to changes in growth state or environmental conditions. The expression of a given gene is determined by contributions from the sequence of the DNA template, regulatory proteins, and reaction conditions. While the input provided by DNA sequence is “hard-coded” (barring mutational change), the inputs provided by regulatory factors and reaction conditions are flexible, enabling them to change in response to alterations in environmental conditions. A multitude of transcription factors and reaction conditions can modulate gene expression. Thus, changes in gene expression occurring in response to alterations in growth state or environmental conditions cannot be predicted a priori from DNA sequence alone. Experiments proposed will enable a systems-level description of the sequence-dependent, factor-dependent, condition- dependent transcriptional landscape in Escherichia coli. Each step of transcription can be rate limiting, and thus serve as a potential target of regulation. During the project period, mechanisms of transcriptional control during initial transcription and elongation, i.e., the stages when RNA synthesis occurs, will be investigated. In future work, studies will expand to include initiation and termination, using similar approaches. A distinguishing feature of the work described has been the development of methods to monitor transcription across extensive sequence space, both in vitro and in vivo. In addition, the flexibility provided by use of E. coli RNAP as a model system, enables straightforward analysis of the contributions of any transcription factor or reaction condition to RNAP activity, both in vitro and in vivo. The results of empirical measurements of RNAP activity across extensive sequence space or across the entire E. coli genome will be integrated into a site- and base-specific quantitative model describing how RNAP activity is specified by inputs from DNA sequence, transcription factors, and conditions. The biological knowledge gained will provide an unprecedently rich description of the architecture of gene expression control in E. coli and provide the basis for extending this approach to other organisms, including eukaryotes.