Abstract
This chapter discusses the biochemical assaying of Gre factors of Thermus Thermophilus. The biochemical studies of Escherichia coli Gre factors indicate that they are not nucleases but RNAP co-factors, which activate the same catalytic center involved in both RNA synthesis and RNA hydrolysis reactions. The biological role of factor-induced endonucleolytic reaction includes: the enhancement of transcription fidelity, by helping RNAP excise misincorporated nucleotides; suppression of transcriptional pausing and arrest by reactivation of RNAP during reversible and irreversible backtracking; and stimulation of RNAP promoter escape and transition from initiation to elongation stage of transcription by helping the catalytic center reengage with nascent RNA 3´-terminus during abortive synthesis. The chapter describes four new methods useful for biochemical and structure-functional studies of Tth Gre factors: direct chromatographic assay for competitive binding of GreA1 and GreA2 to RNAP, specific transcript cleavage (misincorporation–excision) assay for GreA1, inhibition of RNA synthesis assay for GreA2, and localized Fe2 + -induced hydroxyl radical mapping of GreA1 and GreA2 sites proximal to RNAP catalytic center.
Published Version
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