Direct interactions between the coiled-coil tip of DksA and the trigger loop of RNA polymerase mediate transcriptional regulation

摘要

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.