Global regulation of genome transcription by modulation of RNA polymerase function

这项研究的目的是了解4000个基因在大肠杆菌基因组上转录调控的分子基础。此前我们发现，大肠杆菌中的RNA聚合酶核心酶的总数只有每个基因组2000个分子。有限数量的RNA聚合酶在4000个基因中的分布是通过调节RNA聚合酶的启动子选择性来控制的。这种调控涉及7种Sigma亚单位(启动子识别亚单位)和大约300种转录因子。为了揭示RNA聚合酶调控的分子机制，我们进行了以下几方面的研究：1)构建了一种新的启动子检测载体TFP(Two-Follow Protein)，其中插入了约1000个启动子。利用这一启动子收集，通过测量绿色荧光蛋白/绿色荧光蛋白比率的表达，确定了启动子强度的生长相耦合变化。2)以鉴定…更多的基因在每个Sigma或转录因子的控制下，使用E.coliDNA芯片对每个缺乏一个特定因子的大肠杆菌突变体进行微阵列试验。3)鉴定每个转录因子识别的DNA序列，建立了一种改进的SELEX方法，用于体外筛选每个转录因子识别的启动子。4)利用纯化的RNA聚合酶和转录因子在体外分析每个转录因子的转录调控方式，并用微阵列和SELEX鉴定启动子。5)制备针对每个转录因子的抗体，用于测定不同生长条件下每个转录因子和转录因子的细胞内浓度。6)建立NIP(NIP)方法，以确定每个转录因子在大肠杆菌基因组上的体内定位对来自大肠杆菌的所有两个组分的系统蛋白进行了激酶分析。在同源对之间观察到反式磷酸化，但在有限的组合中，非同源感受器激酶观察到反应规则的磷酸化，表明转录因子激活过程中存在串扰。较少

The aim of this research was undertaken to understand the molecular basis of transcription regulation of 4,000 genes on the Escherchia coli genome. Previously we found that the total number of RNA polymerase core enzyme in E coli is only 2,000 molecules per genome. The distribution of a limited number of RNA polymerase among 4,000 genes is controlled through modulation of the promoter selectivity of RNA polymerase. For this modulation, 7 species of the sigma subunit (promoter recognition subunit) and about 300 species of transcription factors are involved. In order to reveal the molecular mechanism of RNA polymerase modulation, we have performed several lines of studies as follows :1)A new promoter assay plasmid vector TFP (two-fluorescent protein) was constructed, into which about 1,000 E. coli promters were inserted. Using this promter eollection, growth phase-coupled variation of the promoter strength has been determined by measuring the expression of GFP/RFP ratio.2)To identify the … More genes under the control of each sigma or transcription factor, the microarray assay was carried out for E. coli mutants, each lacking one specific factor, using E. coli DNA chip.3)To identify the DNA sequences recognized by each transcription factor, an improved SELEX method was developed and applied for in vitro screening promoters recognized by each purified transcription factor.4)The mode of transcription regulation by each transcription factor has been analyzed in vitro using the purified RNA polymerase and transcription factors and the promoters identified by microarray and SELEX.5)Antibodies against each transcription factor were prepared and used for determination of the intracellular concentration of each sigma and transcription factor under various growth conditions.6)NIP (nucleoid immuno-precipitation) method was developed to identify the localization in vivo of each transcription factor on the E. coli genome.7)Kinetics and specificity of regulator phosphorylation by sensor kinase was analyzed for all two-comonent sytem proteins from E. coli. Trans-phosphorylation was observed between cognate pairs but for a limited combinations, the phosphorylation of response regular was observed by non-cognate sensor kinase, indicating the cross-talk in the activation process of transcription factor. Less