Genome-Wide Definition of the SigF Regulon in Mycobacterium tuberculosis
Genome-Wide Definition of the SigF Regulon in Mycobacterium tuberculosis
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DOI：
10.1128/jb.06692-11
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发表时间：
2012-04-01
影响因子：
3.2
通讯作者：
Cole, Stewart T.
中科院分区：
文献类型：
--
作者：
Hartkoorn, Ruben C.;Sala, Claudia;Cole, Stewart T.
在结核分枝杆菌中，替代 sigma 因子 SigF 通过改变 RNA 聚合酶特异性来控制特定基因子集的表达。在这里，我们利用两种全基因组方法来识别 SigF 结合位点：染色质免疫沉淀 (ChIP-on-chip) 和 SigF 介导的转录本的微阵列分析。由于 SigF 在生长的对数期不是丰富的蛋白质，因此使用普那霉素 IA 诱导系统来控制其表达。我们鉴定了 67 个高亲和力 SigF 结合位点和 16 个 SigF 启动子指导转录本表达的位点。这些基因座包括 sigF 本身、参与脂质和中间代谢和毒力的基因，以及至少一种可能在 SigF 下游发挥作用的转录调节因子 (Rv2884)。此外，还发现SigF指导小RNA F6基因的转录。还发现了许多 SigF 可能参与反义转录的位点，并且在两种情况下（Rv1358 和 Rv1870c），SigF 依赖性启动子位于预测的编码序列内。定量 PCR 证实了微阵列的结果，并且使用 cDNA 末端的 5'-快速扩增来绘制 SigF 特异性转录起始点。在 11 个基因之前发现了规范的 SigF 共有启动子序列 GGTTT-N(15-17)-GGGTA。总之，这些数据有助于定义 SigF 调节子，并表明 SigF 不仅控制毒力因子 HbhA 等蛋白质的表达，而且还影响非编码 RNA 和反义转录本等新功能。
In Mycobacterium tuberculosis the alternative sigma factor SigF controls the expression of a particular subset of genes by altering RNA polymerase specificity. Here, we utilize two genome-wide approaches to identify SigF-binding sites: chromatin immunoprecipitation (ChIP-on-chip) and microarray analysis of SigF-mediated transcripts. Since SigF is not an abundant protein in the logarithmic phase of growth, a pristinamyin IA-inducible system was used to control its expression. We identified 67 high-affinity SigF-binding sites and 16 loci where a SigF promoter directs the expression of a transcript. These loci include sigF itself, genes involved in lipid and intermediary metabolism and virulence, and at least one transcriptional regulator (Rv2884), possibly acting downstream of SigF. In addition, SigF was also found to direct the transcription of the gene for small RNA F6. Many loci were also found where SigF may be involved in antisense transcription, and in two cases (Rv1358 and Rv1870c) the SigF-dependent promoter was located within the predicted coding sequence. Quantitative PCR confirmed the microarray findings and 5'-rapid amplification of cDNA ends was used to map the SigF-specific transcriptional start points. A canonical SigF consensus promoter sequence GGTTT-N(15-17)-GGGTA was found prior to 11 genes. Together, these data help to define the SigF regulon and show that SigF not only governs expression of proteins such as the virulence factor, HbhA, but also impacts novel functions, such as noncoding RNAs and antisense transcripts.