Biochemical Assays of Gre Factors of Thermus Thermophilus

Publisher Summary 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.