Control of Transcription Initiation

细菌RNA聚合酶由与σ特异性因子相关的核心（β、β '、2个α和ω）组成。 在大肠在大肠杆菌中，主要的sigma是sigma 70，由rpoD编码。在sigma 70家族中有数百种细菌sigma因子，并且它们都共享同源序列和功能区域。 区域2.4/ 3和区域4.2识别启动子DNA内的特异性序列，分别与延伸的-10和-35启动子元件结合。 间隔区被认为对于设定这些元件之间的距离是重要的，具有有限的序列特异性。 克罗恩病（CD）是一种炎症性肠病，由胃肠道的免疫攻击引起。虽然CD的病因不明，但粘附性侵袭性E。大肠杆菌，如致病菌株LF 82，经常被发现与CD相关，这表明它可能在CD发病机制中起作用。基因组分析表明，LF 82的多个核苷酸替换使其与大肠杆菌和大肠杆菌区别开来。大肠杆菌中，包括rpoD基因序列的改变以编码sigma 70的D445 V变体。奇怪的是，在将LF 82引入改变的Schaedler植物群无菌小鼠（其胃肠道仅被8种细菌物种（缺乏肠杆菌科）定殖）后，rpoD变体（以及其他）迅速回复到大肠杆菌σ 70中更高度保守的D445残基。杆菌 在E. coli K-12遗传背景，我们发现sigma 70 D445 V置换赋予在平板上生长的强冷敏感表型和在低温下的极端粘液样表型。 我们模拟了E. coli RNA聚合酶与启动子DNA的比较表明，σ 70 D445在相对于转录起始位点的位置-17处与非模板链相距5埃。因此，该残基靠近σ 70依赖性启动子的-10和-35元件之间的间隔区，该区域通常不包含特定的序列决定簇。D445也在β ' E42的5埃范围内，β' E42是位于β '-拉链内的残基，据报道与间隔区DNA相互作用。 为了研究sigma 70 D445 V对转录的影响，我们纯化了该变体，并测定了其对核心/sigma 70缔合和在理想的-10/-35启动子处的开放复合物形成的影响。 我们的研究结果表明，突变并不简单地使RNA聚合酶在较冷的温度下有缺陷，这表明它可能会影响某些启动子的转录。 我们对E.大肠杆菌野生型和变异株在培养物中或平板上分别在37 ° C和23 ° C下生长。 在23 ° C或37 ° C的培养物中，对于几个基因，包括ampC（β-内酰胺酶）、mdtK和acrZ（多药外排泵）以及csrD（使小RNA csrB/csrC不稳定），变体的RNA倍数差异显著增加;相对于wt降低的RNA值包括csrC、malT（麦芽糖操纵子的调节子）和putP（脯氨酸摄取）的RNA值。 有趣的是，特别是在37 ° C培养中，参与rpoS翻译激活的dsrA和rprA RNA的相对水平增加，而特别是在23 ° C培养中，参与趋化性和运动性的多个基因的相对水平降低。 我们还表明，D445 V的变体表现出增加的耐氨苄青霉素，但不卡那霉素和利福平，和运动的变体受损，特别是在较低的温度。 我们的研究结果表明，rpoD序列变异可能会影响一组基因的表达，这些基因为致病菌在CD肠道内提供选择性优势，并可能揭示细菌适应环境的新策略。

Bacterial RNA polymerase is composed of a core (beta, beta', 2 alphas, and omega) that associates with the sigma specificity factor. In E. coli the primary sigma is sigma70, encoded by rpoD. There are hundreds of bacterial sigma factors within the sigma 70 family, and they all share regions of homologous sequence and function. Regions 2.4/ 3, and region 4.2 recognize specific sequences within the promoter DNA, binding to the extended -10 and to the -35 promoter elements, respectively. The spacer is thought to be important for setting the distance between these elements, with limited sequence specificity. Crohns disease (CD), an inflammatory bowel disease, arises from an immune attack of the GI tract. Although the etiology of CD is unknown, Adherent Invasive E. coli, such as the pathobiont strain LF82, is frequently found associated with CD, suggesting that it may play a role in CD pathogenesis. Genome analysis of LF82 has revealed multiple nucleotide substitutions that distinguish it from commensal and toxigenic E. coli, including a change in the rpoD gene sequence to encode a D445V variant of the sigma 70. Curiously, after introduction of LF82 into altered Schaedler Flora gnotobiotic mice, whose GI tracts are colonized with only 8 bacterial species (lacking Enterobacteriaceae), the rpoD variant (as well as others) rapidly reverts to the more highly conserved D445 residue in sigma70 of commensal E. coli. After constructing isogenic strains in an E. coli K-12 genetic background, we have found that the sigma70 D445V substitution confers a strong cold sensitive phenotype for growth on plates and an extreme mucoid phenotype at low temperatures. Our modeled structure of E. coli RNA polymerase with promoter DNA indicates that sigma70 D445 is 5 angstroms from the non-template strand at position -17 relative to the transcription start site. Thus, this residue is near the spacer region between the -10 and -35 elements of a sigma70-dependent promoter, a region that does not typically contain specific sequence determinants. D445 is also within 5 angstroms of beta' E42, a residue that lies within the beta'-zipper and has been reported to interact with spacer DNA. To investigate the effect of sigma70 D445V on transcription, we have purified the variant and assayed its effect on core/sigma70 association and open complex formation at an ideal -10/-35 promoter. Our results indicate that the mutation does not simply render RNA polymerase defective at colder temperatures, suggesting that it might affect transcription from certain promoters. We have performed RNA-seq analyses of the E. coli wt and variant strain grown in culture or on plates at 37o C vs. 23o C. In either 23o C or 37o C cultures, the RNA fold-difference for the variant increases significantly for several genes, including ampC (beta-lactamase), mdtK and acrZ (multi-drug efflux pump), and csrD (destabilizes the small RNAs csrB/csrC); lowered RNA values relative to wt include those of csrC, malT (regulator of maltose operon), and putP (proline uptake). Interestingly, specifically in the 37o C culture, the relative levels of dsrA and rprA RNAs, which are involved in the activation of rpoS translation, increase, while specifically in the 23o C culture, the relative levels of multiple genes involved in chemotaxis and motility decrease. We also show that the D445V variant exhibits increased resistance to ampicillin, but not to kanamycin and rifampicin, and that the motility of the variant is impaired especially at lower temperatures. Our results suggest that the rpoD sequence variant may affect the expression of sets of genes that offer the pathobiont a selective advantage within the CD gut, and may reveal a novel strategy for environmental adaptation by bacteria.