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

In lineages like E. coli, (p)ppGpp produced in response to charged-tRNA deficiency 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 stimulate RNAP pausing, inhibit catalysis, or promote backtracking allosterically, may inhibit GTP synthesis, may compete for GTP binding, or may uncouple transcription–translation by inhibiting ribosomes. Using a combination of cryo-EM, biochemical assays, quantitative nascent elongating transcript sequencing (qNET-seq), and quantitative ChIP-seq (qChIP-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.