Activation of transcription at sigma 54-dependent promoters on linear templates requires intrinsic or induced bending of the DNA.
The initiation of transcription (open complex formation) on supercoiled DNA templates carrying the sigma 54-dependent promoters glnAp2 or glnHp2 can be readily activated by NR1-phosphate bound to sites located 100 bp upstream from the transcriptional start site. In the case of glnAp2, open complex formation can also be activated by NR1-phosphate on a linear template, but in the case of glnHp2 activation on a linear template requires in addition to NR1-phosphate, a DNA-bending protein such as the histone-like protein HU or integration host factor (IHF). Moving the binding sites for NR1 200 bp further away from glnHp2 allows transcription to be activated equally well in the absence or presence of HU, and in this case IHF inhibits the open complex formation. Furthermore, replacement of the DNA segment separating the binding sites for NR1 from glnAp2 by a random sequence of bases of equal length, does not reduce open complex formation on supercoiled DNA but prevents open complex formation on linear DNA unless HU is provided. These observations indicate that with binding sites for NR1 located in their usual position, 100 bp from the transcriptional start site, the DNA segment separating these sites from the promoter must be either intrinsically bent or bent by HU or, in the case of glnHp2, by IHF to allow contact between activator and the sigma 54-RNA polymerase-promoter complex. Computer simulation of the shape of the DNA suggests that in the case of glnAp2, but not of glnHp2 or the altered glnAp2, this segment has an intrinsic curvature of 70 degrees.
Carmona
M
M
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
Magasanik
B
B
D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D034982:Integration Host Factors; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic / Q000235:genetics; D051877:RNA Polymerase Sigma 54; D017421:Sequence Analysis; D012808:Sigma Factor / Q000235:genetics*; D015533:Transcriptional Activation / Q000235:genetics*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D034982:Integration Host Factors; D012808:Sigma Factor; C040330:histone-like protein HU, bacteria; C497751:rpoN protein, E coli; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54