Region 3.2 of the σ factor controls the stability of rRNA promoter complexes and potentiates their repression by DksA

Abstract

The σ factor drives promoter recognition by bacterial RNA polymerase (RNAP) and is also essential for later steps of transcription initiation, including RNA priming and promoter escape. Conserved region 3.2 of the primary σ factor (‘σ finger’) directly contacts the template DNA strand in the open promoter complex and facilitates initiating NTP binding in the active center of RNAP. Ribosomal RNA promoters are responsible for most RNA synthesis during exponential growth but should be silenced during the stationary phase to save cell resources. In Escherichia coli, the silencing mainly results from the action of the secondary channel factor DksA, which together with ppGpp binds RNAP and dramatically decreases the stability of intrinsically unstable rRNA promoter complexes. We demonstrate that this switch depends on the σ finger that destabilizes RNAP–promoter interactions. Mutations in the σ finger moderately decrease initiating NTP binding but significantly increase promoter complex stability and reduce DksA affinity to the RNAP–rRNA promoter complex, thus making rRNA transcription less sensitive to DksA/ppGpp both in vitro and in vivo. Thus, destabilization of rRNA promoter complexes by the σ finger makes them a target for robust regulation by the stringent response factors under stress conditions.

Rewiring of growth-dependent transcription regulation by a point mutation in region 1.1 of the housekeeping σ factor

“…Analysis of the in vivo rrnB P1 activity was performed as described in ( 32 ). WT or I48S E. coli strains were transformed with plasmid pET28_rrnBP1_luxCDABE encoding the complete luciferase operon from Photorhabdus luminescence under the control of the rrnB P1 promoter.…”

“…Escherichia coli core RNAP was expressed from the pVS10 plasmid and purified as described previously ( 33 ). Wild-type σ 70 containing an N-terminal His 6 -tag was expressed from the pET28- rpoD vector and purified from inclusion bodies, followed by protein renaturation and Ni-affinity chromatography ( 3 , 32 ). Mutant σ 70 containing the I48S substitution was obtained by site-directed mutagenesis and purified in the same way.…”

“…Mutant σ 70 containing the I48S substitution was obtained by site-directed mutagenesis and purified in the same way. DksA containing an N-terminal His 6 -tag was expressed in E. coli BL21(DE3) from the pET28-DksA plasmid ( 32 ). All buffer solutions used during DksA purification contained 0.1 mM ZnCl 2 .…”

“…Supercoiled pTZ19-based plasmid containing the RNA I promoter (108–110 nt full-length RNA) and rrnB P1 promoter followed by the hisT terminator (88 nt full-length RNA) was obtained as described in ( 32 ). Linear DNA fragments containing the rrnB P1, T7A1cons or λP R promoters ( Supplementary Figure S5 ) were obtained by PCR from synthetic oligonucleotides and plasmid templates ( 3 ).…”

“…The presence of heparin or rifapentin in the nucleotide mixtures was required to prevent transcription re-initiation in control reactions lacking DNA competitors. The data were fitted to a one-exponential equation to determine the observed rate constants ( k obs ) and half-life times ( t 1/2 = ln 2/ k obs ) for dissociation of promoter complexes ( 32 ).…”