Allosteric control of Escherichia coli rRNA promoter complexes by DksA.
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 A away, between positions -6 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.
Rutherford
Steven T
ST
Department of Bacteriology, University of Wisconsin at Madison, Madison, Wisconsin 53706, USA.
Villers
Courtney L
CL
Lee
Jeong-Hyun
JH
Ross
Wilma
W
Gourse
Richard L
RL
D000494:Allosteric Regulation; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006159:Guanosine Tetraphosphate / Q000378:metabolism; D008958:Models, Molecular; D011401:Promoter Regions, Genetic / Q000235:genetics*; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D013489:Suppression, Genetic
D029968:Escherichia coli Proteins; C090751:dksA protein, E coli; D006159:Guanosine Tetraphosphate; D012321:DNA-Directed RNA Polymerases