Global regulation of genome transcription by modulation of RNA polymerase function

这项研究的目的是了解土方大肠杆菌基因组上4,000个基因转录调控的分子基础。此前我们发现，大肠杆菌中RNA聚合酶核心酶的总数量仅为每个基因组2,000个分子。有限数量的RNA聚合酶在4,000个基因中的分布通过调节RNA聚合酶的启动子选择性来控制。对于这种调节，涉及7种sigma亚基（启动子识别亚基）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）构建了一个新的启动子检测质粒载体TFP（two-fluorescent protein），将约1,000个E.插入大肠杆菌启动子。使用这种启动子收集，通过测量GFP/RFP比率的表达来确定启动子强度的生长相偶联变化。 ...更多信息 基因的控制下的每一个西格玛或转录因子，微阵列分析进行了E. coli突变体，每一个都缺少一个特定的因子，使用E. coli DNA chip. 3）为了鉴定每个转录因子识别的DNA序列，4）利用纯化的RNA聚合酶和转录因子，以及基因芯片和SELEX技术鉴定的启动子，对转录因子的转录调控模式进行了体外分析。制备针对每种转录因子的抗体，并用于测定各种生长条件下每种σ和转录因子的细胞内浓度。6）开发NIP（类核免疫沉淀）方法以鉴定每种转录因子在E. 7）对大肠杆菌的所有二元系统蛋白进行了传感激酶磷酸化调节因子的动力学和特异性分析。杆菌在同源对之间观察到反式磷酸化，但对于有限的组合，通过非同源传感器激酶观察到响应规则的磷酸化，表明转录因子激活过程中的串扰。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