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

Abstract

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.

DksA regulates RNA polymerase in Escherichia coli through a network of interactions in the secondary channel that includes Sequence Insertion 1

“…2B). Thirteen DksA-Bpa variants preferentially cross-linked to β', as expected from earlier results that identified β' as a cross-linking partner of DksA (12), and five preferentially cross-linked to β (Fig. 2 B and C).…”

“…2D and SI Appendix, Fig. S3 A-D, identified eight new cross-linking sites and increased the precision of three previously identified sites (DksA F69, E79, and E146) by an order of magnitude (12). DksA-Bpa cross-links mapped to two new regions in β that were not predicted by current models: a region overlapping β-SI1 and one that included a substrate-binding region from β.…”

“…Second, the C-terminal α-helix of DksA extended out toward β-SI1 in the secondary channel. Of the three docking positions, two positions placed the globular domain of DksA closer to the β' jaw domain (β'1147-1245), whereas only one positioned the globular domain of DksA closer to the tip of the β' rim helices, a known binding determinant of DksA (12,16). We used this solution for further refinement of the structural model.…”

Key features of σ S required for specific recognition by Crl, a transcription factor promoting assembly of RNA polymerase holoenzyme

“…The tag was removed with thrombin. BPA-substituted heart muscle kinase (HMK)-His-σ S variants were expressed (42) and purified by Ni-affinity chromatography. Proteins were 32 P-radiolabeled on an N-terminally encoded HMK recognition site using HMK (15,43).…”

“…Proteins were 32 P-radiolabeled on an N-terminally encoded HMK recognition site using HMK (15,43). Photoactivated cross-linking of BPA-substituted proteins was performed in duplicate (42).…”