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  • Title: A direct effect of guanosine tetraphosphate on pausing of Escherichia coli RNA polymerase during RNA chain elongation.
    Author: Kingston RE, Nierman WC, Chamberlin MJ.
    Journal: J Biol Chem; 1981 Mar 25; 256(6):2787-97. PubMed ID: 7009598.
    Abstract:
    The effect of the regulatory nucleotide ppGpp on transcription by Escherichia coli RNA polymerase in vitro has been studied using bacteriophage T7 and T3 DNAs as templates. We have previously described the development of transcriptional test systems using these templates that can sensitively detect changes in promotor or terminator recognition of an RNA polymerase (Wiggs, J.L., Bush, J.W., and Chamberlin, M.J. (1979) Cell 16, 97-109) or changes in the rate of any of the major steps of the transcription cycle (Chamberlin, M.J., Nierman, W.C., Wiggs, J.L., and Neff, N. (1979) J. Biol. Chem. 254, 10061-10069). Using these procedures we fail to detect any substantial alteration by ppGpp of the normal interaction of E. coli RNA polymerase with the several T7 major and minor promoter sites or of the rate of productive RNA chain initiation at either T7 promoter A1 (ATP start) or A2 (GTP start). However, at physiologically relevant concentrations (KI approximately 50 microM), ppGpp significantly lowers the overall elongation rate of T7 RNA chains, leading to a substantial reduction in the overall rate of RNA synthesis. Inhibition of transcriptional elongation does not appear to be competitive with the ribonucleoside triphosphate substrates. Furthermore, ppGpp does not inhibit chain elongation during transcription of the synthetic polynucleotide templates poly[d(A-T)] or poly[d(A-G):d(C-T)]. We conclude that ppGpp interacts directly with some site on RNA polymerase other than one of the sites used for substrate binding. Furthermore, the inhibition must depend on specific DNA sequences present in T7 DNA, but not in poly[d(A-T)] or poly[d(A-G):d(C-T)]. Analysis of T7 transcripts formed in the presence and absence of ppGpp by gel electrophoresis reveals that in the latter instance there is enhanced pausing of the transcriptional elongation complex at specific sites on the template. It is likely that the enzyme also pauses at such sequences in the absence of ppGpp, but for a far briefer time (Kassavetis, G.A., and Chamberlin, M.J. (1981) J. Biol. Chem. 256, 2777-2786). Thus, ppGpp appears to slow transcriptional elongation by binding to RNA polymerase and altering its structure in a manner that impedes passage of the enzyme through certain DNA sequences. The presence of similar transcriptional barriers in rRNA operons activated by the presence of ppGpp could lead selectively to large reductions in the rate of rRNA synthesis in vivo.
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