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.
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.
Rippe
K
K
Deutsches Krebsforschungszentrum Abteilung Biophysik der Makromoleküle, Heidelberg, Germany.
Guthold
M
M
von Hippel
P H
PH
Bustamante
C
C
D001426:Bacterial Proteins / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins; D005974:Glutamate-Ammonia Ligase / Q000235:genetics; D018625:Microscopy, Atomic Force; D051922:PII Nitrogen Regulatory Proteins; D011401:Promoter Regions, Genetic; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000235:genetics*; D015534:Trans-Activators; D014157:Transcription Factors; D015533:Transcriptional Activation
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D051922:PII Nitrogen Regulatory Proteins; D012808:Sigma Factor; D015534:Trans-Activators; D014157:Transcription Factors; C497876:glnG protein, E coli; C497751:rpoN protein, E coli; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54; C081173:glutamine synthetase I; D005974:Glutamate-Ammonia Ligase