Most bacterial transcription factors are DNA-binding proteins that recognize a particular DNA sequence, thereby regulating transcription of nearby promoters. However, the 151-amino-acid (17.5-kDa) DksA protein does not bind to DNA, but rather has been proposed to bind to the RNA polymerase (RNAP) secondary channel (Perederina et al. 2004; Lennon et al. 2009; Rutherford et al. 2009). DksA regulates transcription in conjunction with the small molecules guanosine tetraphosphate and pentaphosphate (referred to here as ppGpp) and the transcript's initiating nucleotide (iNTP) by modifying the kinetic properties of the promoter–RNAP complex (Barker et al. 2001; Paul et al. 2004). Although the concentration of DksA remains relatively constant during growth of Escherichia coli (Paul et al. 2004; Rutherford et al. 2007; Chandrangsu et al. 2011), the concentrations of ppGpp and NTPs fluctuate, accounting for the dynamic effects of DksA on transcriptional responses to changing nutritional conditions (Murray et al. 2003; Paul et al. 2004). DksA and ppGpp together inhibit transcription from rRNA promoters, many ribosomal protein and tRNA promoters, the promoter for FlhDC (the master regulator of flagella synthesis), the Fis promoter, DksA's own promoter, and as many as 300 others and directly activate promoters for amino acid biosynthesis and/or transport, virulence, the sRNA-binding protein Hfq, σE-dependent transcription, and as many as 400 others (for review,see Haugen et al. 2008; see also Potrykus et al. 2006; Magnusson et al. 2007; Durfee et al. 2008; Traxler et al. 2008; Lemke et al. 2009, 2011; Chandrangsu et al. 2011; J Lemke and RL Gourse, unpubl.). However, the mechanisms responsible for inhibition and activation remain ill-defined. Transcription initiation begins with the binding of RNAP holoenzyme (subunit composition α2ββ′ωσ) to promoter DNA to form an initial closed complex (RPC) (Haugen et al. 2008; Saecker et al. 2011). This complex isomerizes to additional intermediates, abbreviated here as RPI, before formation of a transcriptionally competent open complex (RPO) and RNA synthesis (Haugen et al. 2008; Saecker et al. 2011). DksA inhibits the transition from RPC to RPI (Rutherford et al. 2009). We proposed that by binding in the RNAP secondary channel, DksA causes an allosteric change in the bridge helix (BH) and/or the trigger loop (TL) that ultimately affects the switch regions of RNAP. The switches interact with the promoter near the transcription start site and control RNAP clamp opening, thereby affecting DNA contacts further downstream in RPO (Rutherford et al. 2009). Rather than achieving promoter specificity by binding to specific DNA sites, DksA exploits promoter-specific variation in the kinetics of transcription initiation (Paul et al. 2004, 2005; Rutherford et al. 2009). At most promoters, the RNAP–promoter complex is long-lived. However, at rRNA promoters, this complex is intrinsically short-lived, such that dissociation of RNAP from rRNA promoters is in competition with NTP addition (Barker et al. 2001). DksA/ppGpp decrease the lifetime of the RNAP–promoter complex, inhibiting rRNA transcription (Barker et al. 2001; Paul et al. 2004; Haugen et al. 2008), whereas long-lived promoters are not inhibited by DksA/ppGpp because RNAP escapes from the complex before dissociation significantly affects transcriptional output (Barker et al. 2001; Paul et al. 2004, 2005). Although we have a kinetic framework for understanding DksA's effects on transcription initiation (Rutherford et al. 2009) and there is a high-resolution structure of DksA (Perederina et al. 2004), a molecular understanding of its mechanism of action has been hampered by the absence of a structure of a DksA–RNAP complex. Structural information is available for the secondary channel-binding factors GreB, TFIIS, and Gfh1 in complex with RNAP (Kettenberger et al. 2003; Opalka et al. 2003; Tagami et al. 2010). However, these factors primarily affect transcription elongation rather than initiation, and their amino acid sequences bear little resemblance to DksA. Furthermore, a DksA homolog has not been identified in the thermophilic organisms from which all reported bacterial RNAP structures to date have been obtained. An RNAP–DksA complex structure has not been reported, and E. coli DksA does not appear to bind to, or function on, Thermus RNAP (T Gaal, CE Vrentas, and RL Gourse, unpubl.). Here we present a model for the interaction of DksA with E. coli RNAP developed by site-specific incorporation of the cross-linkable amino acid benzoyl-phenylalanine (Bpa) (Chin et al. 2002) into DksA, mapping of cross-linked sites in RNAP, and then computational docking of DksA on RNAP. The model, in conjunction with the results of hydroxyl radical protein–protein footprinting and characterization of variants of RNAP that affect DksA binding and/or function, unambiguously positions DksA in the secondary channel and identifies interactions with both the rim helices (RHs), located at the entrance to the secondary channel, and the TL of the β′ subunit. Engineered cysteine residues in the DksA coiled-coil tip (cc-tip) and the TL resulted in formation of a disulfide bond between them. We propose that a TL–DksA interaction provides an explanation for the sequence requirement of the cc-tip for regulating transcriptional output and plays a central role in the mechanism of DksA action.