NusG Is a Sequence-specific RNA Polymerase Pause Factor That Binds to the Non-template DNA within the Paused Transcription Bubble

NusG, referred to as Spt5 in archaeal and eukaryotic organisms, is the only transcription factor conserved in all three domains of life. This general transcription elongation factor binds to RNA polymerase (RNAP) soon after transcription initiation and dissociation of the RNA polymerase sigma factor. Escherichia coli NusG increases transcription processivity by suppressing RNAP pausing, whereas Bacillus subtilis NusG dramatically stimulates pausing at two sites in the untranslated leader of the trpEDCFBA operon. These two regulatory pause sites participate in transcription attenuation and translational control mechanisms, respectively. Here we report that B. subtilis NusG makes sequence-specific contacts with a T-rich sequence in the non-template DNA (ntDNA) strand within the paused transcription bubble. NusG protects T residues of the recognition sequence from permanganate oxidation, and these T residues increase the affinity of NusG to the elongation complex. Binding of NusG to RNAP does not require interaction with RNA. These results indicate that bound NusG prevents forward movement of RNA polymerase by simultaneously contacting RNAP and the ntDNA strand. Mutational studies indicate that amino acid residues of two short regions within the NusG N-terminal domain are primarily responsible for recognition of the trp operon pause signals. Structural modeling indicates that these two regions are adjacent to each another in the protein. We propose that recognition of specific sequences in the ntDNA and stimulation of RNAP pausing is a conserved function of NusG-like transcription factors.