A sigma54-dependent promoter in the regulatory region of the Escherichia coli rpoH gene.
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 sigma54 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 sigma54-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 sigma54 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 sigma54 promoters, PspF and NtrC, were tested but neither of them acted specifically. Therefore, PspFDeltaHTH, 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 PspFDeltaHTH from a plasmid was employed. Thus, the sigma54-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 sigma54-RNAP-promoter initiation complex was formed in vitro and in vivo and needed only an ATP-dependent activator to start transcription.
Janaszak
Anna
A
Department of Molecular Biology, University of Gdańsk, Kładki 24, 80-822 Gdańsk, Poland.
Majczak
Wiktor
W
Nadratowska
Beata
B
Szalewska-Palasz
Agnieszka
A
Konopa
Grazyna
G
Taylor
Alina
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D051877:RNA Polymerase Sigma 54 / Q000502:physiology*; D012808:Sigma Factor / Q000235:genetics* / Q000378:metabolism; D024363:Transcription Initiation Site; D014158:Transcription, Genetic
D006360:Heat-Shock Proteins; D012808:Sigma Factor; C069774:heat-shock sigma factor 32; D000255:Adenosine Triphosphate; D051877:RNA Polymerase Sigma 54