Purification and assay of Escherichia coli transcript cleavage factors GreA and GreB

Abstract

Publisher Summary This chapter discusses the purification and assaying of Escherichia coli (E. coli) transcript cleavage factors, GreA and GreB. Two homologous prokaryotic proteins, GreA and GreB, isolated from E. coli along with eukaryotic protein SII constitute a novel class of transcription factors that prevent or suppress the elongation arrest, a condition when RNA polymerase (RNAP) is locked in ternary complexes that can neither propagate nor dissociate. In addition to the anti arrest activity, GreA have a proofreading role in transcription because it facilitates the removal of misincorporated nucleotides. Both Gre proteins were originally discovered as transacting factors, present in trace amounts in standard preparations of E. coli RNAP, by virtue of their ability to stimulate cleavage of nascent RNA in ternary elongation complexes (TEC). The reaction constitutes endonucleolytic hydrolysis of the nascent RNA followed by the dissociation of the 3' proximal oligonucleotide fragment (2-18 nucleotides) from TEC and restart of elongation from newly generated 3′-OH terminus. Similar cleavage reactions are described for ternary transcription complexes of vaccinia virus RNAP, and eukaryotic RNAP III and RNAP I, which indicates that the transcript cleavage reaction is an important evolutionarily conserved function.