Determination of the promoter strength in the mixed transcription system: promoters of lactose, tryptophan and ribosomal protein L10 operons from Escherichia coli.
In vitro transcription was performed in a single reaction mixture, which contained three species of truncated E. coli DNA template, each carrying one specific promoter, lacP (UV5), trpP or rplJp, and the transcripts of distinct sizes were analyzed by electrophoresis on the same gel. Using this "mixed transcription" system, the order of the promoter strength, i.e., the capacity to form stable open complex, was determined in the single-round transcription under the standard conditions (50 mM NaCl and 37 degrees C) to be lacP greater than trpP greater than rplJp, the latter two promoters being 30--40% and 5--10% the strength of lacP, respectively. After the multiple-round transcription, however, the level of trp transcription was the lowest due to low cyclic-reaction rate but became the highest when another trp fragment containing the natural terminator was used as the template. The order of the transcription level also varied depending on the ionic strength and the reaction temperature and, as a result, lacP was no more the strongest under high salt concentration and at high temperature.
D001482:Base Composition; D004262:DNA Restriction Enzymes; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D007700:Kinetics; D007785:Lactose / Q000235:genetics*; D009876:Operon; D012333:RNA, Messenger / Q000235:genetics; D000080882:Ribosomal Protein L10; D012269:Ribosomal Proteins / Q000235:genetics*; D012293:Rifampin / Q000494:pharmacology; D013698:Templates, Genetic; D014158:Transcription, Genetic / Q000187:drug effects; D014364:Tryptophan / Q000235:genetics*
D012333:RNA, Messenger; D012269:Ribosomal Proteins; D014364:Tryptophan; D012321:DNA-Directed RNA Polymerases; D004262:DNA Restriction Enzymes; D007785:Lactose; D012293:Rifampin