The ribosome is the central component of protein synthesis in all cells. In E. coli, the rate of ribosomal RNA synthesis determines the rate of ribosome synthesis. Multiple mechanisms contribute to the expression of rRNA. A cis-acting sequence (the UP Element) and a trans-acting protein (FIS) account for the high activity of rRNA promoters. In addition, at least two regulatory factors, ppGpp and an unidentified feedback regulator, account for the stringent control and growth rate dependent control of rRNA transcription, respectively. In the next funding period, experiments are proposed to determine the mechanism by which the known activators and regulators affect RNA polymerase and to identify the remaining effectors that influence rRNA transcription. These studies have broad implications not only for our understanding of ribosome synthesis in bacteria, but also for our understanding of basic transcription mechanisms in prokaryotes and eukaryotes. The molecular details of the interactions between the components involved in transcription activation of rrn P1 promoters (RNAP, the UP Element, and FIS) will be determined. Recognition of the UP Element by RNAP represents a new paradigm in our picture of bacterial transcription. The study of FIS-RNAP interactions is also of interest, since FIS- dependent activation represents an activation class unlike those characterized previously. Thus, the identification of the critical residues in RNAP affecting UP Element and FIS utilization and the development of an UP Element consensus sequence should be major contributions to our understanding of transcription. Potential roles of both the alpha and sigma subunits of RNAP will be investigated. Both genetic and biochemical approaches to these problems are described. Compensating effects between multiple regulators have obscured the ability to determine the responses of individual systems to specific environmental signals affecting rRNA transcription. The cis-acting targets for all the known mechanisms affecting transcription from rrnB P1 have now been identified. By utilizing promoter mutations which selectively eliminate promoter responses to FIS, ppGpp, or feedback, it should be possible to determine the contribution of FIS-dependent activation to rRNA expression at specific times, the mechanism of transcription inhibition by ppGpp, and the molecular identity of the feedback regulator.
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