The Escherichia coli DksA protein inserts into the RNA polymerase (RNAP) secondary channel, modifying the transcription initiation complex so that promoters with specific kinetic characteristics are regulated by changes in the concentrations of ppGpp and NTPs. We used footprinting assays to determine the specific kinetic intermediate, RP I , on which DksA acts. Genetic approaches identified substitutions in the RNAP switch regions, bridge helix, and trigger loop that mimicked, reduced, or enhanced DksA function on rRNA promoters. Our results indicate that DksA binding in the secondary channel of RP I disrupts interactions with promoter DNA at least 25 Å away, between positions À6 and +6 (the transcription start site is +1). We propose a working model in which the trigger loop and bridge helix transmit effects of DksA to the switch region(s), allosterically affecting switch residues that control clamp opening/closing and/or that interact directly with promoter DNA. DksA thus inhibits the transition to RP I . Our results illustrate in mechanistic terms how transcription factors can regulate initiation promoter-specifically without interacting directly with DNA.[Keywords: RNA polymerase; promoter; DksA; ppGpp; transcription initiation; ribosome synthesis] Supplemental material is available at http://www.genesdev.org. DksA, ppGpp, and NTPs work together to regulate rRNA synthesis in Escherichia coli (Paul et al. 2004). DksA concentrations are relatively constant (Rutherford et al. 2007), but ppGpp and NTP concentrations vary dramatically with nutrient availability (Murray et al. 2003). Inactivation of the dksA gene derepresses rRNA transcription, uncoupling ribosome production from the cellular demand for protein synthesis, because direct modification of RNA polymerase (RNAP) by DksA is needed for changes in the concentrations of ppGpp and NTPs to exert effects on the transcription initiation complex (Paul et al. 2004).The mechanism of DksA action remains unclear. Unlike conventional regulators of transcription initiation, DksA does not bind to DNA but instead interacts directly with RNAP (Paul et al. 2004;Perederina et al. 2004). Biochemical studies and structural similarities between DksA and the transcription elongation factors GreA and GreB suggest that DksA binds in the RNAP secondary channel (Opalka et al. 2003;Perederina et al. 2004; S.T. Rutherford, I. Toulokhonov, C.E. Vrentas, W. Ross, and R.L. Gourse, unpubl.), but there is no structure of DksA bound to RNAP, and the precise interactions between RNAP and DksA have yet to be defined.Because DksA binds RNAP instead of a specific DNA sequence, it has the potential to affect all promoter complexes. Consistent with this prediction, DksA decreases the lifetimes of complexes formed by all promoters tested to date (Paul et al. 2004(Paul et al. , 2005Rutherford et al. 2007). However, DksA directly affects transcriptional output only from a subset of promoters, including many needed for the synthesis of ribosomes, virulence, membrane stress responses, and amino aci...