Bacterial RNA polymerase (RNAP) relies on the same active site for RNA synthesis and co-transcriptional RNA proofreading. The intrinsic RNA proofreading activity of RNAP can be greatly stimulated by Gre factors, which bind within the secondary channel and directly participate in the RNA cleavage reaction in the active site of RNAP. Here, we characterize mutations in Escherichia coli RNAP that differentially affect intrinsic and Gre-stimulated RNA cleavage. Substitution of a highly conserved arginine residue that contacts nascent RNA upstream of the active site strongly impairs intrinsic and GreA-dependent cleavage, without reducing GreA affinity or catalytic Mg2+ binding. In contrast, substitutions of several nonconserved residues at the Gre-interacting interface in the secondary channel primarily affect GreB-dependent cleavage, by decreasing both the catalytic rate and GreB affinity. The results suggest that RNAP residues not directly involved in contacts with the reacting RNA groups or catalytic ions play essential roles in RNA cleavage and can modulate its regulation by transcription factors. (C) 2019 Elsevier Inc. All rights reserved.