σ54-Promoter Discrimination and Regulation by ppGpp and DksA

Abstract

The 54 -factor controls expression of a variety of genes in response to environmental cues. Much previous work has implicated the nucleotide alarmone ppGpp and its co-factor DksA in control of 54 -dependent transcription in the gut commensal Escherichia coli, which has evolved to live under very different environmental conditions than Pseudomonas putida. Here we compared ppGpp/DksA mediated control of 54 -dependent transcription in these two organisms. Our in vivo experiments employed P. putida mutants and manipulations of factors implicated in ppGpp/DksA mediated control of 54 -dependent transcription in combination with a series of 54 -promoters with graded affinities for 54 -RNA polymerase. For in vitro analysis we used a P. putida-based reconstituted 54 -transcription assay system in conjunction with DNA-binding plasmon resonance analysis of native and heterologous 54 -RNA polymerase holoenzymes. In comparison with E. coli, ppGpp/DksA responsive 54 -transcription in the environmentally adaptable P. putida was found to be more robust under low energy conditions that occur upon nutrient depletion. The mechanism behind this difference can be traced to reduced promoter discrimination of low affinity 54 -promoters that is conferred by the strong DNA binding properties of the P. putida 54 -RNA polymerase holoenzyme.To ensure promoter specificity, the bacterial multisubunit catalytic core RNA polymerase (RNAP) 4 (␣ 2 ␤␤Ј) is guided to the distinct promoter classes within the genome by a dissociable -factor. In addition to the household 70 -factor, most bacteria encode a variable number of alternative -factors that fall into two families based on primary amino acid sequences and regulatory properties. The largest of these is the 70 -family, which encompasses most alternative -factors within four subgroups that all form RNAP holoenzymes that can spontaneously initiate transcription (1). Orthologs of the structurally distinct 54 -subunit are the only members of the second family. 54 imposes kinetic constrains that lock the 54 -RNAP holoenzyme in a non-productive closed complex. Thus, 54 -dependent transcription always requires activators, called bacterial enhancer-binding proteins or bEBPs, which use ATP catalysis to remodel 54

Regulation of Alternative Sigma Factor Use

“…Consistent with the idea that Rsd could facilitate access of alternative sigmas to core-RNAP, the naturally elevated levels of Rsd in stationary-phase E. coli sequester a significant portion (∼25%) of σ 70 (72). However, it should be emphasized that Rsd null mutants have minimal effects on σ 38 -and σ 54 -dependent promoter outputs that are enhanced by overexpression of Rsd (9,64), suggesting that Rsd does not act alone to bring about these regulatory events. An intriguing finding from structural studies of the Rsd/σ 4 complex is that a network of interactions connects the binding interface with other potential binding cavities located on the surface of Rsd.…”

“…Nevertheless, a σ S promoter that is not dependent directly on ppGpp still requires ppGpp for activity in vivo even when reduced σ S levels are compensated for by ectopic expression (46, 52). Likewise, although the levels of σ 54 are constant irrespective of the presence or absence of ppGpp and/or DksA, the activities of σ 54 promoters that are not enhanced directly by either factor in vitro are still greatly stimulated by the presence of these molecules in vivo (9,10,53,84). These findings demand an alternative explanation for the action of ppGpp.…”

“…DksA and ppGpp can have mutually independent and sometimes opposing effects (1, 3, 75, and references therein); however, DksA sensitizes RNAP to the cellular levels of ppGpp to account for their common coaction (70). In E. coli and P. putida, DksA levels are relatively constant (9,75); therefore, it is the changing levels of ppGpp-the herald of stress-that instigate proactive promoterspecific and global transcriptional responses that allow the cell to prepare for tough times ahead.…”

Inter-sigmulon communication through topological promoter coupling

“…Native P. putida KT2440 core RNAP, σ 70 -RNAP holoenzyme, σ 54 , His-DksA and the constitutively active ΔA2-His-DmpR protein were purified as previously described (31,33,34,38). …”

“…In contrast to σ 70 - and other σ 70 -like RNAP holoenzymes that can spontaneously initiate transcription, σ 54 -RNAP forms thermodynamically stable closed promoter complexes and so strictly requires activation by a member of a specialized family of mechano-transcriptional activators (reviewed in 29). The σ 70 -Pr promoter controls the levels of DmpR—the obligate (methyl)phenol-responsive mechano-transcriptional activator of the powerful σ 54 -Po promoter, which drives transcription of the genes for the specialised catabolic enzymes (30–33). In contrast to the σ 54 -Po promoter, the σ 70 -Pr promoter is intrinsically weak and requires the co-action of ppGpp and DksA to overcome constraints imposed by poor binding of σ 70 -RNAP and a slow rate of open-complex formation (34–36).…”