SUMMARY Throughout the bacterial domain, the alarmone ppGpp dramatically reprograms transcription following nutrient limitation. This “stringent response” is critical for survival and antibiotic-tolerance and is a model for transcriptional regulation by small ligands. We report that ppGpp binds to two distinct sites 60 angstroms apart on E. coli RNA polymerase (RNAP), one characterized previously (Site 1) and a second identified here at an interface of RNAP and the transcription factor DksA (Site 2). The location and unusual tripartite nature of Site 2 account for the DksA-ppGpp synergism and suggest mechanisms for ppGpp enhancement of DksA’s effects on RNAP. Site 2 binding results in the majority of ppGpp’s effects on transcription initiation in vitro and in vivo, and strains lacking Site 2 are severely impaired for growth following nutritional shifts. Filling of the two sites at different ppGpp concentrations would expand the dynamic range of cellular responses to changes in ppGpp levels.

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...

Escherichia coli DksA is a transcription factor that binds to RNA polymerase (RNAP) without binding to DNA, destabilizing RNAP-promoter interactions, sensitizing RNAP to the global regulator ppGpp, and regulating transcription of several hundred target genes, including those encoding rRNA. Previously, we described promoter sequences and kinetic properties that account for DksA's promoter specificity, but how DksA exerts its effects on RNAP has remained unclear. To better understand DksA's mechanism of action, we incorporated benzoylphenylalanine at specific positions in DksA and mapped its cross-links to RNAP, constraining computational docking of the two proteins. The resulting evidence-based model of the DksA-RNAP complex as well as additional genetic and biochemical approaches confirmed that DksA binds to the RNAP secondary channel, defined the orientation of DksA in the channel, and predicted a network of DksA interactions with RNAP that includes the rim helices and the mobile trigger loop (TL) domain. Engineered cysteine substitutions in the TL and DksA coiledcoil tip generated a disulfide bond between them, and the interacting residues were absolutely required for DksA function. We suggest that DksA traps the TL in a conformation that destabilizes promoter complexes, an interaction explaining the requirement for the DksA tip and its effects on transcription.

E. coli DksA works in conjunction with the small molecule ppGpp to regulate transcription initiation negatively or positively, depending on the identity of the promoter. DksA is in a class of transcription factors that do not bind directly to DNA like classical repressors or activators but rather bind in the RNA polymerase (RNAP) secondary channel like the transcription elongation factors GreA and GreB in E. coli and TFIIS in eukaryotes. We found that substitution for either of two residues in its coiled-coil tip, D74 or A76, eliminates DksA function without affecting its apparent affinity for RNAP. The properties of DksA-Gre factor chimeras indicated that the coiled-coil tip is responsible for the DksA-specific effects on open complex formation. A conservative substitution at position 74, D74E, resulted in a loss of DksA function in both negative and positive control, and an E44D substitution at the analogous position in GreA resulted in a gain of function in both negative and positive control. That a single methylene group has such an extraordinary effect on these transcription factors highlights the critical nature of the identity of coiled-coil tip interactions with RNAP for open complex formation.

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