Publisher Summary Two homologous prokaryotic proteins, GreA and GreB, L2 and a eukaryotic analog TFIIS (SII) comprise a novel class of transcription factors that are involved in endonucleolytic hydrolysis of nascent RNA in ternary complexes (TCs) of RNA polymerase (RNAP). Unlike classic RNases, SII and Gre proteins do not display any nucleolytic activity toward free RNA or RNA-DNA duplex and generally require formation of a stable TC. In the absence of factors, the intrinsic endonucleolytic activity of Escherichia coli RNAP and eukaryotic RNAP II can be stimulated by mild alkaline pH 5 and pyrophosphate, respectively. Both RNA synthesis and RNA hydrolysis reactions require at least two Mg 2+ ions with similar K m values. Moreover, the two reactions are activated and inhibited by similar groups of divalent ions, such as Mn 2+ , Fe 2+ , and Co 2+ , and Pb 2+ , Cu 2+ , and Zn 2+ , respectively. These results suggest that the same active center of RNAP is involved in both reactions. The transcript cleavage occurs when RNAP in a TC engages in “backtracking,” that is characterized by reverse translocation of RNAP together with the transcription bubble along the DNA template. Backtracking has been demonstrated both in vitro and in vivo ; however, the nature of the signals that cause it (besides destabilization of the DNA-RNA hybrid in the TC) is poorly understood.