Specificity of promoter site utilization in vitro by bacterial RNA polymerases on Bacillus phage phi 29 DNA. Transcription mapping with exonuclease III.
Bacillus subtilis RNA polymerase holoenzyme transcribes phi 29 DNA in vitro producing five major RNA species defined by characteristic electrophoretic mobilities. In addition to these products, Escherichia coli RNA polymerase transcribes phi 29 DNA to yield three RNA species not detected when transcribing with the B. subtilis enzyme under the same optimal reaction conditions for RNA synthesis. Transcriptional analysis of purified restriction fragments and exonuclease III-digested DNA established locations of six promoter and three termination sites defining the eight transcripts. The transcription map shows that E. coli RNA polymerase initiates transcription at three sites not efficiently utilized by the B. subtilis enzyme. However, initiation by the B. subtilis polymerase from at least two of these sites could be detected at E:DNA ratios greater than 10 in the absence of competing promoters. These results indicate that differences between the two polymerases in promoter utilization are not explained by specificity of promoter binding, but represent differences in responding to promoter strength. Transcription of phi 29 DNA and T7 DNA by E. coli core polymerase with either B. subtilis or E. coli sigma subunits results in formation of transcripts identical with those produced by E. coli holoenzyme, suggesting that core polymerase contains elements important in determining relative promoter strength. The efficiency of rifampicin-resistant complex formation on phi 29 and T7 promoters is also dependent upon the source of core polymerase.
D001412:Bacillus subtilis / Q000201:enzymology*; D001435:Bacteriophages / Q000378:metabolism*; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005090:Exodeoxyribonucleases; D005092:Exonucleases / Q000378:metabolism*; D046911:Macromolecular Substances; D008970:Molecular Weight; D009876:Operon; D012293:Rifampin / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects
D004279:DNA, Viral; D046911:Macromolecular Substances; D012321:DNA-Directed RNA Polymerases; D003851:Deoxyribonucleases; D005090:Exodeoxyribonucleases; D005092:Exonucleases; C020133:exodeoxyribonuclease III; D004262:DNA Restriction Enzymes; D012293:Rifampin