Abstract
DksA controls transcription of genes associated with diverse stress responses, such as amino acid and carbon starvation, oxidative stress, and iron starvation. DksA binds within the secondary channel of RNA polymerase, extending its long coiled-coil domain towards the active site. The cellular expression of DksA remains constant due to a negative feedback autoregulation, raising the question of whether DksA activity is directly modulated during stress. Here, we show that Escherichia coli DksA is essential for survival in acidic conditions and that, while its cellular levels do not change significantly, DksA activity and binding to RNA polymerase are increased at lower pH, with a concomitant decrease in its stability. NMR data reveal pH-dependent structural changes centered at the interface of the N and C-terminal regions of DksA. Consistently, we show that a partial deletion of the N-terminal region and substitutions of a histidine 39 residue at the domain interface abolish pH sensitivity in vitro. Together, these data suggest that DksA responds to changes in pH by shifting between alternate conformations, in which competing interactions between the N- and C-terminal regions modify the protein activity.
Highlights
Escherichia coli DksA has been shown to play a key role in regulation of transcription of ribosomal RNA and protein genes [1,2] and may contribute to genome integrity by preventing conflicts between replication and transcription machineries [3]
A hypothesis that pH-dependent conformational switches in the secondary channel regulators may play a role in adaptation to pH stress, suggested by a conformational change observed in Gfh1 upon pH downshift [21], is consistent with earlier reports that S. typhimurium and S. flexneri ΔdksA mutants are sensitive to low pH [8,27]
We show that DksA activity is stimulated by a pH downshift and that this change correlates with a stronger affinity for RNA polymerase (RNAP)
Summary
Escherichia coli DksA has been shown to play a key role in regulation of transcription of ribosomal RNA and protein genes [1,2] and may contribute to genome integrity by preventing conflicts between replication and transcription machineries [3]. DksA, often in synergy with the alarmone ppGpp, controls expression of a large number of genes required for motility [4,5], fimbriae biogenesis [6], pathogenesis [7,8], and stress responses to very diverse cellular signals, ranging from nutrient limitation [2] to oxidative and nitrosative damage [9]. PpGpp and DksA frequently function synergistically, examples of differential and even opposite regulation continue to accumulate [4,10,11]. Most strikingly, while both ppGpp and DksA are required for rrnB P1 regulation by many cellular signals [1,12], ppGpp is dispensable during phosphate starvation [10].
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