PMID:9236116
| Citation |
Rippe, K, Guthold, M, von Hippel, PH and Bustamante, C (1997) Transcriptional activation via DNA-looping: visualization of intermediates in the activation pathway of E. coli RNA polymerase x sigma 54 holoenzyme by scanning force microscopy. J. Mol. Biol. 270:125-38 |
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| Abstract |
Scanning force microscopy (SFM) has been used to study transcriptional activation of Escherichia coli RNA polymerase x sigma 54 (RNAP x sigma 54) at the glnA promoter by the constitutive mutant NtrC(D54E,S160F) of the NtrC Protein (nitrogen regulatory protein C). DNA-protein complexes were deposited on mica and images were recorded in air. The DNA template was a 726 bp linear fragment with two NtrC binding sites located at the end and about 460 bp away from the RNAP x sigma 54 glnA promoter. By choosing appropriate conditions the structure of various intermediates in the transcription process could be visualized and analyzed: (1) different multimeric complexes of NtrC(D54E,S160F) dimers bound to the DNA template; (2) the closed complex of RNAP x sigma 54 at the glnA promoter; (3) association between DNA bound RNAP x sigma 54 and NtrC(D54E,S160F) with the intervening DNA looped out; and (4) the activated open promoter complex of RNAP x sigma 54. Measurements of the DNA bending angle of RNAP x sigma 54 closed promoter complexes yielded an apparent bending angle of 49(+/-24) degrees. Under conditions that allowed the formation of the open promoter complex, the distribution of bending angles displayed two peaks at 50(+/-24) degrees and 114(+/-18) degrees, suggesting that the transition from the RNAP x sigma 54 closed complex to the open complex is accompanied by an increase of the DNA bending angle. |
| Links | |
| Keywords |
Bacterial Proteins/metabolism; DNA, Bacterial/genetics; DNA-Binding Proteins/metabolism; DNA-Directed RNA Polymerases/genetics; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins; Glutamate-Ammonia Ligase/genetics; Microscopy, Atomic Force; PII Nitrogen Regulatory Proteins; Promoter Regions, Genetic; RNA Polymerase Sigma 54; Sigma Factor/genetics; Trans-Activators; Transcription Factors; Transcriptional Activation |
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