Efficient anchoring of RNA polymerase in Escherichia coli during coupled transcription-translation of genes encoding integral inner membrane polypeptides.
While it has been known that supercoiling of the DNA template can be induced by transcription, the mechanism and the efficiency of this process in vivo is not fully understood. We report here that transcription of genes encoding 16 S rRNA, a stable RNA species, or cytoplasmic polypeptides leads to very little or no detectable DNA supercoiling even under the optimum conditions in Escherichia coli. This indicates that hydrodynamic drag on the transcription complex (including RNA polymerase, nascent RNA, ribosomes, and nascent polypeptides) is not sufficient to anchor RNA polymerase during coupled transcription-translation. On the other hand, transcription of membrane-associated genes encoding integral inner membrane or exported periplasmic polypeptides leads to apparent DNA supercoiling. Transcription of genes encoding integral inner membrane polypeptides leads to significantly greater anchoring of RNA polymerase than does transcription of genes encoding periplasmic polypeptides. This may reflect differences in the coupling of transcription-translation with membrane association during expression of these two classes of polypeptides. Evidence is further presented to suggest that the anchoring of RNA polymerase is probably achieved through the interaction of nascent polypeptides with the cytoplasmic surface of the inner membrane during coupled transcription-translation. Moreover, transcriptions of a membrane-associated gene can, under certain circumstances, induce topological anchoring of an RNA polymerase transcribing a neighboring gene that ordinarily is not membrane-associated. Finally, the potential biological consequences of our findings are discussed.
Ma
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Department of Chemistry, University of California, Berkeley.
Cook
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Pon
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Hearst
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D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry; D004278:DNA, Superhelical / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010455:Peptides / Q000235:genetics; D010957:Plasmids; D014176:Protein Biosynthesis; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004278:DNA, Superhelical; D008565:Membrane Proteins; D010455:Peptides; D012321:DNA-Directed RNA Polymerases