Thompson Karl M, Rhodius Virgil A, Gottesman Susan
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
J Bacteriol. 2007 Jun;189(11):4243-56. doi: 10.1128/JB.00020-07. Epub 2007 Apr 6.
RybB is a small, Hfq-binding noncoding RNA originally identified in a screen of conserved intergenic regions in Escherichia coli. Fusions of the rybB promoter to lacZ were used to screen plasmid genomic libraries and genomic transposon mutants for regulators of rybB expression. A number of plasmids, including some carrying rybB, negatively regulated the fusion. An insertion in the rep helicase and one upstream of dnaK decreased expression of the fusion. Multicopy suppressors of these insertions led to identification of two plasmids that stimulated the fusion. One contained the gene for the response regulator OmpR; the second contained mipA, encoding a murein hydrolase. The involvement of MipA and OmpR in cell surface synthesis suggested that the rybB promoter might be dependent on sigma(E). The sequence upstream of the +1 of rybB contains a consensus sigma(E) promoter. The activity of rybB-lacZ was increased in cells lacking the RseA anti-sigma factor and when sigma(E) was overproduced from a heterologous promoter. The activity of rybB-lacZ and the detection of RybB were totally abolished in an rpoE-null strain. In vitro, sigma(E) efficiently transcribes from this promoter. Both a rybB mutation and an hfq mutation significantly increased expression of both rybB-lacZ and rpoE-lacZ fusions, consistent with negative regulation of the sigma(E) response by RybB and other small RNAs. Based on the plasmid screens, NsrR, a repressor sensitive to nitric oxide, was also found to negatively regulate sigma(E)-dependent promoters in an RseA-independent fashion.
RybB是一种小的、与Hfq结合的非编码RNA，最初是在对大肠杆菌保守基因间区域的筛选中鉴定出来的。RybB启动子与lacZ的融合用于筛选质粒基因组文库和基因组转座子突变体，以寻找RybB表达的调控因子。许多质粒，包括一些携带RybB的质粒，对该融合有负调控作用。rep解旋酶中的一个插入以及dnaK上游的一个插入降低了融合基因的表达。这些插入的多拷贝抑制子导致鉴定出两个刺激该融合的质粒。一个含有应答调节因子OmpR的基因；另一个含有编码胞壁质水解酶的mipA。MipA和OmpR参与细胞表面合成表明RybB启动子可能依赖于σ(E)。RybB +1上游的序列包含一个一致的σ(E)启动子。在缺乏RseA抗σ因子的细胞中以及当从异源启动子过量产生σ(E)时，RybB-lacZ的活性增加。在rpoE缺失的菌株中，RybB-lacZ的活性和RybB的检测完全消失。在体外，σ(E)能有效地从该启动子转录。RybB突变和hfq突变均显著增加了RybB-lacZ和rpoE-lacZ融合基因的表达，这与RybB和其他小RNA对σ(E)应答的负调控一致。基于质粒筛选，还发现对一氧化氮敏感的阻遏物NsrR以不依赖RseA的方式对σ(E)依赖的启动子进行负调控。
