Rho is a termination factor and an ATPase that binds to nascent RNA transcript and catalyzes their site-specific release within many gram- negative bacterial and phage transcription units. This proposal outlines a research program for the construction and characterization of a paused transcription intermediate that can be used as a substrate to analyze the thermodynamics and kinetics of the Rho-mediated transcript release reaction. The transcription template will be constructed from a segment of DNA containing the E. coli rho attenuator (tRA), a Rho-dependent terminator that is located in front of the rho gene, and which autogenously regulates the level of Rho in the bacterial cell. To create the paused transcription intermediate, site-directed mutagenesis will be used to install an EcoR1 restriction site at a specific location downstream of the region coding for the Rho-attachment site on the transcript; transcription elongation will be blocked by the binding of a cleavage-defective EcoR1 endonuclease. A kinetic formalism will be used to ascertain the rate-determining steps of the Rho attachment/transcript release reaction. This formalism will be used to evaluate the rates of Rho assembly and transcript release in an effort to sort out the mechanisms of these processes during transcription termination. The kinetics of release and the structure of the Rho-bound ternary complexes and the released products will be characterized in the presence of ATP and ATP analogs to determine how the Rho-ATPase activity is involved in the release process. The location of the EcoR1 blockade will be varied by linker scanning mutagenesis. The relationship between the site of the blockade and the energetics and kinetics of Rho attachment and transcript release will be measured. These studies will provide information on the minimum size for the Rho attachment site, and the significance of sequence context in transcript-template interactions during termination.
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