sigma factor mutations affecting the sequence-selective interaction of RNA polymerase with -10 region single-stranded DNA.

Thesigmasubunit of RNA polymerase determines promoter recognition and catalyzes DNA strand separation. The -35 promoter region is recognized by a helix-turn-helix motif in region 4, while the -10 region is specified, at least in part, by an amphipathic helix in region 2. We have proposed that conserved aromatic residues insigmaregion 2.3 interact with the non-template strand of the -10 element to drive open complex formation. We now report that Bacillus subtilis sigmaA holoenzyme, but neither core nor sigmaA alone, binds with high selectivity to single-stranded (ss) DNA containing the non-template -10 consensus sequence. UV irradiation of holoenzyme-ssDNA complexes efficiently crosslinks sigmaA to DNA and protease mapping supports a primary contact site in or near region 2. Several mutations in sigmaA region 2.3, shown previously to impair promoter melting, affect ssDNA binding: Y184A decreases binding selectivity, while Y189A and W193A decrease the efficiency of photocrosslinking. These results support a model in which these aromatic amino acids are juxtaposed to ssDNA, consistent with their demonstrated role in stabilizing the open complex.     

Sigma38 (rpoS) RNA polymerase promoter engagement via -10 region nucleotides

Band shift assays using DNA probes that mimic closed and open complexes were used to explore the determinants of promoter recognition by sigma38 (rpoS) RNA polymerase. Duplex recognition was found to be much weaker than that observed in sigma70 promoter usage. However, binding to fork junction probes, which attempt to mimic melted DNA, was very strong. This binding occurs via the non-template strand with the identity of the two conserved junction nucleotides (-12T and -11A) being of paramount importance. A modified promoter consensus sequence identified these two nucleotides as among only four (underlined) that are highly conserved, and all four were in the -10 region (CTAcacT from -13 to -7). The remaining two nucleotides were shown to have different roles, -13C in preventing recognition by the heterologous sigma70 polymerase and -7T in directing enzyme isomerization. These -10 region nucleotides appear to have their primary function prior to full melting because probes that had a melted start site were relatively insensitive to substitution at these positions. These results suggest the sigma38 mechanism differs from the sigma70 mechanism, and this difference likely contributes to selective use of sigma38 under conditions that exist during stationery phase.     

真核基因启动子非依赖的功能转录延伸复合物的体外组装

真核生物RNA聚合酶Ⅱ的持续合成能力对基因转录过程中每一个阶段,包括启动子脱离、转录暂停、转录终止以及转录偶联DNA损伤修复过程的调节至关重要.在RNA聚合酶Ⅱ介导的转录延伸过程中,其和模板DNA及转录产物RNA紧密结合,形成一个非常稳定的延伸三维复合物(elongationcomplex,EC).此特征性“泡”状结构的形成是RNA聚合酶Ⅱ持续合成能力所必需的.在不依赖启动子及众多转录起始因子的条件下,利用人工合成的RNA与DNA寡核苷酸,在体外组装形成具有功能转录活性的延伸复合物.结果表明,长度为9个核苷酸的RNA与模板DNA形成的杂合分子对转录延伸复合物的形成是必需的,而非转录模板DNA链的加入导致最终活性转录“泡”状复合物的形成,并可转录形成与模板相关的转录产物,进一步通过在模板DNA的特定位置引入一个乙酰氧乙酰氨基芴修饰基团,可特异性地阻断转录延伸过程,从而显示该系统在研究真核基因转录及转录偶联DNA损伤修复机制中的潜在应用价值.