21 amino acid peptide Microcin J25 (MccJ25) inhibits transcription by bacterial RNA polymerase (RNAP). MccJ25-resistance mutations cluster in the RNAP secondary channel through which incoming NTP substrates are thought to reach the catalytic center and the 3' end of the nascent RNA is likely to thread in backtracked transcription complexes. The secondary channel also accepts transcript cleavage factors GreA and GreB. Here, we demonstrate that MccJ25 inhibits GreA/GreB-dependent transcript cleavage, impedes formation of backtracked complexes, and can be crosslinked to the 3'-end of the nascent RNA in elongation complexes. These results place the MccJ25 binding site within the secondary channel. Moreover, single-molecule assays reveal that MccJ25 binding to a transcribing RNAP temporarily stops transcript elongation but has no effect on the elongation velocity between pauses. Kinetic analysis of single-molecule data allows us to put forward a model of transcription inhibition by MccJ25 that envisions the complete occlusion of the secondary channel by bound inhibitor.