Min Y N, Tabuchi A, Womble D D, Rownd R H
Department of Cell, Molecular, and Structural Biology, Medical School, Northwestern University, Chicago, Illinois 60611.
J Bacteriol. 1991 Apr;173(7):2378-84. doi: 10.1128/jb.173.7.2378-2384.1991.
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.
FII 型质粒 NR1 的稳定性（stb）位点由一个位于两个串联基因上游的必需顺式作用 DNA 位点组成，这两个串联基因编码必需的稳定性蛋白。发现 stb 位点是从第一个基因 stbA 上游的启动子位点转录而来的。该启动子在体内和体外均具有转录活性，且位于包含必需顺式作用位点的区域内。从该位点起始的转录本长度约为 1500 至 1600 个核苷酸。Northern（RNA）印迹分析表明，转录本跨越了 stbA 和下游基因 stbB。缺失启动子的突变体无法从 stbA 或 stbB 产生可检测到的转录本。在相反 DNA 链上的 stbB 基因中包含的第三个开放阅读框 stbC 的转录无法检测到。对于一个 stbA 中插入了转座子的突变体，未检测到 stbB 的转录。在删除大部分转座子后，stbA 中留下了一个 35bp 的移码插入，尽管该插入对 stbB 功能仍有极性效应，但 stbB 的转录得以恢复。通过 RNA 的脉冲标记随后进行定量 RNA-DNA 杂交来测量 stb 位点的体内转录速率。缺失 stbB 的突变体转录速率增加了约 10 倍，而缺失启动子区域的突变体转录速率至少降低了 10 倍。stb mRNA 的半衰期约为 2 分钟。这些数据表明，stbA 和 stbB 作为一个可能进行自动调节的操纵子被共转录。
