Functional Differentiation of Transcription Apparatus

基因组总基因转录的全球调控是基因组测序完成后的研究前沿之一。十年前，我们发现，RNA聚合酶分子总数(约2000个)少于大肠杆菌基因组上的基因总数(约4000个)。基于这一发现，我们认为RNA聚合酶选择基因在给定的环境条件下转录，并且RNA聚合酶的特异性和活性的调节在不同条件下转录模式的转换中起主要作用。这项研究项目就是为了揭示这一模式。本研究的主要结果如下：1)将大肠杆菌RNA聚合酶分两步功能分化为不同形式的转录装置。在第一步中，核心酶与西格玛亚基的七个分子物种中的一个相关联。我们已经测定了所有七个sigma su…的细胞内浓度在不同培养条件下更多的球蛋白，以及每个西格玛亚基与核心酶的结合亲和力。根据这两个值，我们成功地估计了七种全酶的细胞内浓度，每个全酶携带一个特定的西格玛因子。2)使用ONA芯片分析了大肠杆菌突变体的转录模式，每个突变体都缺乏一个西格玛因子。在转录组分析的基础上，我们估计了每个sigma亚基控制的基因。3)RNA聚合酶全酶通过与总共266个转录因子中的一个或两个相互作用而进一步分化。为了鉴定每个转录因子控制下的基因，对缺乏一个特定转录因子的大肠杆菌突变体进行了转录组和蛋白质组分析。4)为了验证转录因子功能的预测，正在使用纯化的RNA聚合酶和转录因子进行候选启动子的体外转录实验。5)针对纯化的转录因子制备了特异性抗体，并正在测定每个转录因子的胞内浓度。6)利用Percoll梯度离心法建立了将大肠杆菌固定相培养成均一细胞群的方法。在分析了分离细胞群体的基因表达模式和转录机制的变化后，我们提出了一个用于大肠杆菌静止相适应的序贯分化模型。较少

Global regulation of transcription of total genes on the genome is one of the research front after completion of the genome sequencing. A decade ago we found that the total number of RNA polymerase molecules (about 2000) is less than the total number of genes (about 4000) on the E. coli genome. Based on this finding, we proposed that the RNA polymerase chooses the genes to transcribe under a given environmental condition, and the modulation of specificity and activity of RNA polymerase plays a major role in switching transcription pattern upon expose to different conditions. This research project has been undertaken to reveal this model. Followings are the major results obtained in this study:1)E. coli RNA polymerase is functionally differentiated into various forms of transcription apparatus in two steps. In the first-step, the core enzyme associates with one of seven molecular species of the sigma subunit. We have determined both the intracellular concentrations of all seven sigma su … More bunits under various culture conditions and the binding affinity of each sigma subunit to the core enzyme. From these two values, we succeeded to estimate the intracellular concentration of seven holoenzymes, each carrying one specific sigma factor.2) Transcription patterns were analyzed, using ONA chips, for E coli mutants, each lacking one sigma factor. Based on the transcriptome analysis, we estimated the genes under the control of each sigma subunit.3) RNA polymerase holoenzyme is further differentiated by interaction with one (or two in a few cases) of a total 266 transcription factors. For identification of genes under the control of each transcription factor, transcriptome and proteome analyses have been carried out for E coli mutants, each lacking one specific transcription factor.4) To examine the prediction for transcription factor functions, in vitro transcription assay of the candidate promoters is being carried out using purified RNA polymerase and transcription factors.5) Specific antibodies have been made against purified transcription factors and the determination of the intracellular concentrations of each transcription factor is in progress.6) Fractionation method of stationary-phase cultures of E. coli into homogenous cell populations has been established by using Percoll gradient centrifugation. After analyses of changes in gene expression pattern and of transcription apparatus for fractionated cell populations, we propose a sequential differentiation model for the stationary-phase adaptation of E coli. Less