Allosteric control of Escherichia coli rRNA promoter complexes by DksA

Abstract

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...

Differential stringent control of Escherichia coli rRNA promoters: effects of ppGpp, DksA and the initiating nucleotides

“…2b). The results support recent findings that DksA is essential for the ppGppdependent inhibition of the rRNA promoters (Rutherford et al, 2009). Moreover, the lack of DksA not only abrogates the inhibitory effect of high ppGpp levels but in addition must somehow affect the basal activity of the rrn promoters during exponential growth.…”

“…This regulation involves the small effector nucleotide ppGpp and the RNA polymerase secondary channel-binding protein DksA (Potrykus & Cashel, 2008;Rutherford et al, 2009;Wagner, 2010). Encouraged by our earlier observation that individual E. coli rRNAs are not transcribed at the same rate and apparently respond differentially to transcriptional regulators, such as FIS, H-NS and LRP (Hillebrand et al, 2005;Liebig & Wagner, 1995;Pul et al, 2005), we asked if the induction of the stringent control might also affect the individual rRNA promoters to a different extent, resulting in non-equal distribution of rRNAs derived from different operons under variable growth conditions.…”

Direct regulation of Escherichia coli ribosomal protein promoters by the transcription factors ppGpp and DksA

“…Although the mRNA sequence signals needed for translational repression are not present in our constructs, and thus translational feedback could not be responsible for the observed in vivo-in vitro discrepancy, perhaps additional factors affected by the ΔdksA mutation contribute to regulation of some r-protein promoters in vivo. (11,36). The promoter sequences that contribute to the susceptibility of rRNA promoter complexes to inhibition by ppGpp/DksA are complex.…”

Transfer RNA instability as a stress response inEscherichia coli: Rapid dynamics of the tRNA pool as a function of demand

“…Nutrient limitation triggers alterations in the activity and promoter preferences of RNA polymerase, and major changes in the cell's gene expression program prime the cell for maintenance and survival rather than growth. 58 Several starvation-regulated factors can directly influence transcription by RNA polymerase, including the alarmone ppGpp (binding at the β′-ω and the β′-DksA interfaces of RNA polymerase), 59,60 the small RNA 6S (binding the σ 70 -RNA polymerase holoenzyme) 61,62 and a number of protein factors (DksA, 63 alternative sigma factors, 64 and anti-sigma factors 65 ) (reviewed in ref. 66).…”