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Half-Site DNA Sequence and Spacing Length Contributions to PrrA Binding to PrrA Site 2 of RSP3361 in Rhodobacter sphaeroides 2.4.1▿ §

机译:半球形DNA序列和间距长度对球形红球菌2.4.1§RSP3361的PrrA位点2的PrrA结合的贡献

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摘要

The consensus DNA binding sequence for PrrA, a global regulator in Rhodobacter sphaeroides 2.4.1, is poorly defined. We have performed mutational analysis of PrrA site 2, of the RSP3361 gene, to which PrrA binds in vitro (J. M. Eraso and S. Kaplan, J. Bacteriol. 191:4341-4352, 2009), to further define the consensus sequence for DNA binding. Two half-sites of equal length, containing 6 nucleotides each, were required for PrrA binding to this DNA sequence. Systematic nucleotide substitutions in both inverted half-sites led to a decrease in binding affinity of phosphorylated PrrA in vitro, the level of which was dependent on the substitution. The reduced binding affinities were confirmed by competition experiments and led to proportional decreases in the expression of lacZ transcriptional fusions to the RSP3361 gene in vivo. The 5-nucleotide spacer region between the half-sites was found to be optimal for PrrA binding to the wild-type half-sites, as shown by decreased PrrA DNA binding affinities to synthetic DNA sequences without spacer regions or with spacer regions ranging from 1 to 10 nucleotides. The synthetic spacer region alleles also showed decreased gene expression in vivo when analyzed using lacZ transcriptional fusions. We have studied three additional DNA sequences to which PrrA binds in vitro. They are located in the regulatory regions of genes positively regulated by PrrA and contain spacer regions with 5 or 8 nucleotides. We demonstrate that PrrA can bind in vitro to DNA sequences with different lengths in the spacer regions between the half-sites.
机译:球形红球菌2.4.1中的全局调节子PrrA的共有DNA结合序列定义不清。我们已经对RSP3361基因的PrrA位点2进行了突变分析,体外与PrrA结合(JM Eraso和S. Kaplan,J. Bacteriol。191:4341-4352,2009),以进一步定义DNA的共有序列捆绑。将PrrA结合到该DNA序列需要两个相等长度的半位,每个半位含有6个核苷酸。在两个反向半位中的系统核苷酸取代导致磷酸化的PrrA的体外结合亲和力降低,其水平取决于取代。竞争实验证实了结合亲和力的降低,并导致lacZ转录融合蛋白在体内与RSP3361基因的表达成比例下降。发现半位点之间的5个核苷酸间隔区对于PrrA与野生型半位点的结合是最佳的,这表现为PrrA DNA与没有间隔区或间隔区范围为1的合成DNA序列的亲和力降低到10个核苷酸。当使用lacZ转录融合体进行分析时,合成的间隔区等位基因在体内也显示出降低的基因表达。我们已经研究了PrrA在体外结合的三个其他DNA序列。它们位于由PrrA阳性调控的基因的调控区中,并包含具有5个或8个核苷酸的间隔区。我们证明PrrA可以在体外结合到DNA序列具有不同长度的半位点之间的间隔区中。

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