ppGpp regulates transcription elongation via direct and indirect inputs to RNA polymerase pausing and nucleotide addition

, (p)ppGpp produced in response to charged-tRNA deficiency directly targets transcribing RNAP polymerase (RNAP) to match its pace to the pioneering ribosome on the nascent RNA (transcription-translation coupling). However, the mechanism by which (p)ppGpp slows RNAP is poorly defined. (p)ppGpp may allosterically stimulate RNAP pausing, inhibit catalysis, promote backtracking, compete for substrate GTP, inhibit GTP synthesis, or uncouple transcription-translation by inhibiting translation. Using a combination of cryo-EM, biochemical assays, and quantitative nascent elongating transcript sequencing (qNET-seq), we establish that (p)ppGpp allosterically regulates pausing and nucleotide addition via distinct motions of the RNAP swivel module and both competes with and lowers GTP in vivo. (p)ppGpp stimulates swiveling at pause sites to delay escape but may also inhibit counter-swiveling required in every round of nucleotide addition. Highlights: ppGpp biases RNAP toward swiveling and away from a catalytically-competent stateppGpp effects on RNAP conformation explain ppGpp stimulation of RNAP pausingppGpp stimulation of RNAP pausing in vivo is mediated mainly by reduced GTP levelsppGpp may allosterically slow RNAP and increases gene occupancy in vivo.