Protein-induced DNA bending clarifies the architectural organization of the sigma54-dependent glnAp2 promoter.
Sigma54-RNA polymerase (Esigma54) predominantly contacts one face of the DNA helix in the closed promoter complex, and interacts with the upstream enhancer-bound activator via DNA looping. Up to date, the precise face of Esigma54 that contacts the activator to convert the closed complex to an open one remains unclear. By introducing protein-induced DNA bends at precise locations between upstream enhancer sequences and the core promoter of the sigma54-dependent glnAp2 promoter without changing the distance in-between, we observed a strong enhanced or decreased promoter activity, especially on linear DNA templates in vitro. The relative positioning and orientations of Esigma54, DNA bending protein and enhancer-bound activator on linear DNA were determined by in vitro footprinting analysis. Intriguingly, the locations from which the DNA bending protein exerted its optimal stimulatory effects were all found on the opposite face of the DNA helix compared with the DNA bound Esigma54 in the closed complex. Therefore, these results provide evidence that the activator must approach the Esigma54 closed complexes from the unbound face of the promoter DNA helix to catalyse open complex formation. This proposal is further supported by the modelling of activator-promoter DNA-Esigma54 complex.
Huo
Yi-Xin
YX
National Laboratory of Protein Engineering and Plant Genetic Engineering, College of life Sciences, Peking University, Beijing 100871, China.
Tian
Zhe-Xian
ZX
Rappas
Mathieu
M
Wen
Jin
J
Chen
Yan-Cheng
YC
You
Cong-Hui
CH
Zhang
Xiaodong
X
Buck
Martin
M
Wang
Yi-Ping
YP
Kolb
Annie
A
D001483:Base Sequence; D002373:Cyclic AMP Receptor Protein; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000737:chemistry*; D003850:Deoxyribonuclease I / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005974:Glutamate-Ammonia Ligase / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D051922:PII Nitrogen Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D051877:RNA Polymerase Sigma 54 / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D002373:Cyclic AMP Receptor Protein; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D051922:PII Nitrogen Regulatory Proteins; D011956:Receptors, Cell Surface; D014157:Transcription Factors; C481662:crp protein, E coli; C497876:glnG protein, E coli; D051877:RNA Polymerase Sigma 54; D003850:Deoxyribonuclease I; C081173:glutamine synthetase I; D005974:Glutamate-Ammonia Ligase