Effects of ppGpp on transcription by DNA-dependent RNA polymerase from Escherichia coli: circular dichroism, absorption and specific transcription studies.
Concrete evidence is presented for conformational changes elicited in RNA polymerase upon binding ppGpp by circular dichroism measurements. In the presence of 100 microM ppGpp, the molar ellipticity of RNA polymerase at 220 nm is reduced by 14% from the initial value of - 11,100 deg X cm2 X dmol-1 at 25 degrees C. In vitro transcription on templates containing the beta-lactamase promoter and colicin E1 promoter on poly[d(A-T)] is inhibited by ppGpp. None of these templates had GC-rich nucleotide sequence near the transcription initiation site, and yet they were influenced by ppGpp. Comparison of the effect on the synthesis of mRNAs for beta-lactamase and colicin E1 and the synthesis of the proteins themselves indicates that the effect of ppGpp is at the level of transcription for the former case and involves coupled transcription-translation for the latter case. Difference absorption, polyacrylamide gel electrophoresis, and nitrocellulose filter-binding studies show that the binding of ppGpp to RNA polymerase does not impair the extent of the interaction between enzyme and DNA. Kinetic studies suggest that ppGpp affects transcription initiation on beta-lactamase promoter. On poly[d(A-T)], ppGpp affects the rate of open complex formation and is a mixed inhibitor with respect to the incorporation of nucleotides. Our results are consistent with the idea that ppGpp acts as a regulator by binding at a site different from the active site and changes the RNA polymerase conformation, causing altered transcriptional behavior on different DNA templates.
Woody
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Woody
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Malcolm
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D002942:Circular Dichroism; D003087:Colicins / Q000235:genetics; D004247:DNA / Q000502:physiology; D004268:DNA-Binding Proteins / Q000502:physiology; D012321:DNA-Directed RNA Polymerases / Q000502:physiology*; D004926:Escherichia coli / Q000502:physiology; D005786:Gene Expression Regulation; D006150:Guanine Nucleotides / Q000502:physiology*; D006159:Guanosine Tetraphosphate / Q000502:physiology*; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D013057:Spectrum Analysis; D014158:Transcription, Genetic; D001618:beta-Lactamases / Q000235:genetics
D003087:Colicins; D004268:DNA-Binding Proteins; D006150:Guanine Nucleotides; D006159:Guanosine Tetraphosphate; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D001618:beta-Lactamases