Allosteric effector ppGpp potentiates the inhibition of transcript initiation by DksA

DksA and ppGpp are the central players in the Escherichia coli stringent response and mediate a complete reprogramming of the transcriptome from one optimized for rapid growth to one adapted for survival during nutrient limitation. A major component of the response is a reduction in ribosome synthesis, which is accomplished by the synergistic action of DksA and ppGpp bound to RNA polymerase (RNAP) inhibiting transcription of rRNAs. Here, we report the X-ray crystal structures of E. coli RNAP holoenzyme in complex with DksA alone and with ppGpp. The structures show that DksA accesses the template strand at the active site and the downstream DNA binding site of RNAP simultaneously and reveal that binding of the allosteric effector ppGpp reshapes the RNAP-DksA complex. The structural data support a model for transcriptional inhibition in which ppGpp potentiates the destabilization of open complexes on rRNA promoters by DksA. We also determined the structure of RNAP-TraR complex, which reveals the mechanism of ppGpp-independent transcription inhibition by TraR. This work establishes new ground for understanding the pleiotropic effects of DksA and ppGpp on transcriptional regulation in proteobacteria.\n\nHighlightsO_LIDksA has two modes of binding to RNA polymerase\nC_LIO_LIDksA is capable of inhibiting the catalysis and influences the DNA binding of RNAP\nC_LIO_LIppGpp acts as an allosteric effector of DksA function\nC_LIO_LIppGpp stabilizes DksA in a more functionally important binding mode\nC_LI