Structure & Function of Prokaryotic Transcript Cleavage

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DESCRIPTION (Provided by applicant): The prokaryotic transcript cleavage factors GreA and GreB identified in Escherichia coli are thought to have three biologically important and evolutionarily conserved functions in transcription: suppression of elongation arrest, facilitation of promoter escape, and enhancement of transcription fidelity. These functions are accomplished by the ability of Gre factors to induce cleavage of nascent RNA in the ternary transcription complex. The broad goal of this project is to understand the mechanism of action and the structure-function relationships of GreA and GreB. Four types of experiments will be carried out. #1. To identify functionally important localities of Gre factors, we will introduce by oligonucleotide directed random mutagenesis single amino acid substitution of all residues in Gre proteins except those that are involved in the intramolecular interactions. We will also introduce single and multiple amino acid substitutions in the conserved loop and in the region immediately preceding the C-terminal domain by site-directed mutagenesis. The mutants will be characterized by specific transcription assays in vivo and in vitro. #2. To elucidate interactions between Gre and other components of the TC, we will identify mutations in the beta and beta prime subunits of RNAP that suppress the lethal phenotypes of dominant negative mutant Gre factors or overproduction of wt Gre. The mutant RNAPs will be purified, and characterized by in vitro transcription assays. #3. Interactions between Gre proteins and RNA polymerase will be explored using Fe2+-induced hydroxyl radical footprinting and mapping, and specific cysteine-directed protein-protein photochemical cross-linking. #4. To obtain three-dimensional structural information, we will prepare crystals of covalently trapped quaternary complex consisting of GreA, DNA template, RNA primer, and the Thermus thermophilus core RNA polymerase and subject them to X-ray crystallographic analysis.