A mutant sigma 32 with a small deletion in conserved region 3 of sigma has reduced affinity for core RNA polymerase

Abstract

.70, encoded by rpoD, is the major v factor in Escherichia coli. rpoD285 (rpoD800) is a small deletion mutation in rpoD that confers a temperature-sensitive growth phenotype because the mutant (o70 is rapidly degraded at high temperature. Extragenic mutations which reduce the rate of degradation of RpoD285 &70 permit growth at high temperature. One class of such suppressors is located in rpoH, the gene encoding &2, an alternative cr factor required for transcription of the heat shock genes. One of these, rpoH113, is incompatible with rpoD+. We determined the mechanism of incompatibility. Although RpoH113 &32 continues to be made when wild-type &70 is present, cells show reduced ability to express heat shock genes and to transcribe from heat shock promoters. Glycerol gradient fractionation of &2 into the holoenzyme and free sigma suggests that RpoH113 &2 has a lower binding affinity for core RNA polymerase than does wild-type 2 . The presence of wild-type a70 exacerbates this defect. We suggest that the reduced ability of RpoH113 &2 to compete with wild-type a70 for core RNA polymerase explains the incompatibility between rpoH113 and rpoD+. The rpoH113 cells would have reduced amounts of -2 holoenzyme and thus be unable to express sufficient amounts of the essential heat shock proteins to maintain viability.Transcription in Escherichia coli is carried out by RNA polymerase, which exists in two forms: core RNA polymerase (a%2, 13, 1'; or E), which carries out elongation and termination, and holoenzyme (a2, 3, 1', C; or Ea), which carries out specific initiation at promoter regions of the DNA (2). E. coli contains multiple sigma factors, and the specificity of promoter binding is determined by the particular a subunit associated with the holoenzyme (for reviews, see references 19 and 37). One way to regulate global gene expression is to modulate the interaction of different a factors with core RNA polymerase in response to physiological and environmental changes (19,26,37). a70, the major a factor in E. coli, is encoded by rpoD (12,18,31) and is essential for cell growth (17,25,35). The rpoD285 mutation and the genetically identical rpoD800 mutation confer a temperature-sensitive (Ts) growth phenotype (17,20,25,30,35) because this mutant sigma factor is unstable and rapidly degraded at high temperature (12, 13). Structural changes in the mutant sigma factor resulting from the 42-bp in-frame deletion in the rpoD285 (rpoD800) allele presumably lead to its instability in vivo (13,20) and in vitro (27). One class of extragenic suppressors of rpoD285 is located in rpoH (htpR), the gene which encodes a32 (14,32,33,45 likely that such a study could provide some insight into how various sigma factors interact in the cell to modulate transcription. Our results indicate that the mutant a32 is present in cells expressing wild-type a70 but is unable to promote transcription from heat shock promoters, probably because it is ineffective in competing with wild-type &7 for binding to core RNA polymerase. This would lead to diminis...

Insights into transcriptional regulation and σ competition from an equilibrium model of RNA polymerase binding to DNA

“…Previously, the number of 70 (29) was thought to be lower than the number of nonelongating E (34), although there was a great deal of evidence for competition (7)(8)(9)(10)14). Our new estimates and careful consideration of the previously published data (18,20,23,24,29,31,35) indicate that 70 and thus total s exceed nonelongating E, and our model rationalizes this finding: this condition is necessary for competition.…”

“…By changing the relative levels of the s, Escherichia coli is thought to coordinate its transcriptional program with growth conditions (11)(12)(13). This view is based on observations indicating that (i) overexpressing one decreases expression of genes controlled by another (7), (ii) mutationally altering binding constants of one for E alters expression by another (14), and (iii) physiological effectors such as ppGpp may act by altering relative binding of s to E (8)(9)(10). In the present work, we use an equilibrium model of RNAP binding to DNA to explore in vivo scenarios that permit transcription regulation by activator recruitment of RNAP and competition.…”

Regulation of Pap Phase Variation

“…RpoD800 contains a 14-amino acid in frame deletion of residues 330 -343 of 70 , which causes it to become unstable at temperatures above 37°C (30). Although the levels of RpoD800 appear to be comparable to wild type RpoD at the permissive temperature, RpoD800 appears to have a lower affinity for core RNA polymerase at the permissive temperature, which could result in a lower level of RNA polymerase-70 holoenzyme (31). Transcription of papBA may be more sensitive to a lowered RNA polymerase level than other genes since it may compete with Lrp for binding near GATC prox .…”

The alarmone (p)ppGpp mediates physiological‐responsive control at the σ54‐dependent Po promoter

“…However, previous work on these mutants provides a plausible mechanistic explanation. These two mutations map in the conserved region 3.1 of sigma 70 -like proteins, which has been implicated by work on rpoH encoding 32 as being important in sigmacore RNAP interaction (Zhou et al, 1992 by the presence of ppGpp (Hernandez and Cashel, 1995 . The growth phase dependency of restoration of transcription from Po in the absence of (p)ppGpp by the RpoD mutant proteins may reflect their mimicry of the action of ppGpp by favouring the association of core with 54 over that with 70 .…”