Transcription elongation, the process of copying DNA into RNA by the RNA polymerase (RNAP), is a crucial point of gene regulation in all organisms. Elongation is not a monotonic process and can be interrupted by pauses, arrests, roadblocks, and termination signals. These events are subject to modulation by various factors that control the rate of RNA product formation. The long-term objective of the proposed work is to determine the basic mechanisms that maintain the proper rate, processivity, and accuracy of transcription in living bacteria cells.
Specific aims of this proposal are: 1. Elucidation of the mechanisms that control the rate and processivity of transcription in E. coli. Preliminary studies demonstrate that the rate of transcription elongation of structural and stable RNA genes depends on growth rate and various stress conditions and is regulated by novel mechanisms. Experiments are proposed to characterize these mechanisms in detail and understand their role in cell adaptation to environmental changes. 2. Elucidation of the mechanism that controls the ? cycle in E. coli. Recent studies indicate that a population of RNAP from E. coli retains initiation-specific ? factor (?70) throughout elongation. The relative amount of this population depends on cellular growth and reaches its maximum during stationary phase. Moreover, the ?-retaining population has a substantially higher ability to support multiple rounds of transcription at certain promoters, suggesting its significant role in gene regulation. Experiments are proposed to determine the molecular mechanism, that controls the release of ?70 and other a factors during elongation. 3. Elucidation of the proofreading mechanism of transcription elongation in E. coli. In vivo, error rates for E. coli RNAP have been estimated to be ~l/1.2x105. However, the determinants of such a high accuracy are unknown. Preliminary data indicates that an unidentified factor exists in E. coli that activates the intrinsic 3'-5' exonuclease (proofreading) activity of RNAP. Experiments are proposed to isolate and characterize this factor in vitro and determine its role in maintaining transcription fidelity in vivo. It is further proposed to examine whether GreA/GreB transcript cleavage factors contribute to transcription fidelity in vivo.
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