A σ 54-dependent promoter in the regulatory region of the Escherichia coli rpoH gene

Abstract

The Escherichia coli rpoH gene is transcribed from four known and differently regulated promoters: P1, P3, P4 and P5. This study demonstrates that the conserved consensus sequence of the s 54 promoter in the regulatory region of the rpoH gene, described previously, is a functional promoter, P6. The evidence for this conclusion is: (i) the specific binding of the s 54 -RNAP holoenzyme to P6, (ii) the location of the transcription start site at the predicted position (C, 30 nt upstream of ATG) and (iii) the dependence of transcription on s 54 and on an ATP-dependent activator. Nitrogen starvation, heat shock, ethanol and CCCP treatment did not activate transcription from P6 under the conditions examined. Two activators of s 54 promoters, PspF and NtrC, were tested but neither of them acted specifically. Therefore, PspFDHTH, a derivative of PspF, devoid of DNA binding capability but retaining its ATPase activity, was used for transcription in vitro, taking advantage of the relaxed specificity of ATP-dependent activators acting in solution. In experiments in vivo overexpression of PspFDHTH from a plasmid was employed. Thus, the s 54 -dependent transcription capability of the P6 promoter was demonstrated both in vivo and in vitro, although the specific conditions inducing initiation of the transcription remain to be elucidated. The results clearly indicate that the closed s 54 -RNAP-promoter initiation complex was formed in vitro and in vivo and needed only an ATP-dependent activator to start transcription.

Use of a promiscuous, constitutively-active bacterial enhancer-binding protein to define the σ54 (RpoN) regulon of Salmonella Typhimurium LT2

“…The heat shock sigma factor gene rpoH has been shown to be under the control of additional promoters and other regulatory proteins in E . coli [36]. The conservation of this promoter region for rpoH in S .…”

“…This is likely due to the presence of σ 70 -type promoters in the cloned sequence. In addition to the σ 54 dependent promoters, other promoters have been reported upstream of glmY , glmZ , rpoH , and yeaG [28,36,41]. …”

The Rhizobium etli RpoH1 and RpoH2 sigma factors are involved in different stress responses

“…There are five promoters upstream of the rpoH gene, recognized by s 70 (P1, P4 and P5), s E (P3) and s 54 (P6) (Erickson et al, 1987;Janaszak et al, 2007). In R. etli, upstream of the rpoH1 gene, two promoter sequences have been identified.…”

Transcription regulation of the Escherichia coli pcnB gene coding for poly(A) polymerase I: roles of ppGpp, DksA and sigma factors

“…DksA protein, and RNA polymerase core and various σ factors were purified according to previously reported procedures (Gamer et al 1992; Janaszak et al 2007; Szalewska-Palasz et al 2007a). The ΔHTH-PspF protein was purified as already described (Jovanovic et al 1999).…”

“…The primer extension reactions were performed essentially as described by Janaszak et al (2007). Briefly, either 50 μg of total RNA or 1.5 μg of specific pcnB mRNA (generated by in vitro transcription), was mixed with 0.5 pmol of 32 P-labeled primer.…”