Universally conserved factors from NusG family bind at the upstream fork
junction of transcription elongation complexes and modulate RNA
synthesis in response to translation, processing, and folding of the
nascent RNA. Escherichia coli NusG enhances transcription elongation in vitro by a poorly understood mechanism. Here we report that E. coli
NusG slows Gre factor-stimulated cleavage of the nascent RNA, but does
not measurably change the rates of single nucleotide addition and
translocation by a non-paused RNA polymerase. We demonstrate that NusG
slows RNA cleavage by inhibiting backtracking. This activity is
abolished by mismatches in the upstream DNA and is independent of the
gate and rudder loops, but is partially dependent on the lid loop. Our
comprehensive mapping of the upstream fork junction by base analogue
fluorescence and nucleic acids crosslinking suggests that NusG inhibits
backtracking by stabilizing the minimal transcription bubble.