Transcription of the stability operon of IncFII plasmid NR1.
The stability (stb) locus of IncFII plasmid NR1 is composed of an essential cis-acting DNA site located upstream from two tandem genes that encode essential stability proteins. The stb locus was found to be transcribed from a promoter site just upstream from the first gene, stbA. This promoter was active for transcription both in vivo and in vitro and was located within the region that includes the essential cis-acting site. Transcripts initiated from this site were approximately 1,500 to 1,600 nucleotides in length. Northern (RNA) blot analysis indicated that the transcripts traversed both stbA and the downstream gene, stbB. Mutants from which the promoter had been deleted failed to produce detectable transcripts from either stbA or stbB. Transcription of a third open reading frame, stbC, which is contained within the stbB gene in the opposite DNA strand, could not be detected. For a mutant in which a transposon had been inserted in stbA, no transcription of stbB was detected. After deletion of most of the transposon, which left behind a 35-bp frameshift insertion in stbA, transcription of stbB was restored, although the insertion still had a polar effect on stbB function. The rate of in vivo transcription of the stb locus was measured by pulse-labeling of RNA followed by quantitative RNA-DNA hybridization. Mutants deleted of stbB had an approximately 10-fold increase in the rate of transcription, whereas those deleted of the promoter region had at least a 10-fold reduction in transcription rate. The half-life of stb mRNA was approximately 2 min. These data suggest that stbA and stbB are cotranscribed as an operon that may be autoregulated.
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Department of Cell, Molecular, and Structural Biology, Medical School, Northwestern University, Chicago, Illinois 60611.
Tabuchi
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Womble
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Rownd
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D015152:Blotting, Northern; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D066298:In Vitro Techniques; D009876:Operon; D010957:Plasmids; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012333:RNA, Messenger