Analysis of the Functional Domains of RNA Polymerase

大肠杆菌依赖DNA的RNA聚合酶由α、β、β‘和Sigma四个主要亚基组成，不仅具有催化RNA合成的活性，而且具有复杂的遗传信息传递功能。为了了解这些功能的结构基础，我们对大肠杆菌RNA聚合酶每个亚基上的功能域进行了系统的分析，得到了以下结果：1)α亚基在两个大的亚基β和β‘组装成酶复合体的过程中起着至关重要的作用。N-末端区域是亚单位组装所必需的，而C-末端区域是转录激活所必需的，位置因子与启动子-35信号上游的DNA结合。然而，由C末端截短的α亚基组成的突变的RNA聚合酶可以被与与启动子-35区域重叠的靶点结合的阳性因子激活。2)已经在β亚基上定位了四个功能位点，包括利福平结合位点、ppGpp敏感性控制位点、启动子选择位点和西格玛结合位点。3)利用针对具有各种西格玛亚基共有的共同氨基酸序列的合成肽的抗体，已经鉴定出可能的新的西格玛亚基。根据这些氨基酸序列设计的DNA探针正在克隆编码这些蛋白质的基因。4)这些基本亚基的功能通过与辅助转录因子的相互作用或酶亚基的修饰来调节。Omega亚单位是与ppGpp相互作用所必需的，ppGpp是一种严格控制的调节分子。5)新型修饰形式的RNA聚合酶全酶存在于固定相的大肠杆菌细胞中，每一种都识别一组不同的启动子。这些结果表明，基因转录的全局控制主要通过调节RNA聚合酶的功能进行。

The DNA-dependent RNA polymerase of Escherichia coli is composed of four major subunits, alpha, beta, beta', and sigma, and carries not only the catalytic activity of RNA synthesis but also complex functions of transmission of genetic information. In order to understand the structural bases for these multiple functions, we carried out a systematic analysis of functional domains on each subunit of E. coli RNA polymerase and obtained the following results.1) The alpha subunit plays an essential role in the assembly of two large subunits, beta and beta', into enzyme complex. The N-terminal region is required for the sub unit assembly, while the C-terminal domain is needed for transcription activation by position factors which bind DNA upstream of the promoter -35 signal. Mutant RNA polymerases consisting of C-terminal truncated alpha subunits are , however, activated by positive factors which bind to target sites that overlap the promoter -35 region.2) Four functional sites have been mapped on the beta subunit, including rifampicin bindint site, ppGpp sensitivity control site, promoter selection site and sigma binding site.3) Putative novel sigma subunits have been identified, using antibodies against synthetic peptides with consensus amino acid sequences common among various sigma subunits. Genes coding for these proteins are being cloned using DNA probes designed based on these amino acid sequences.4) The functions of these basic subunits are modulated by interaction with accessory transcription factors or modification of enzyme subunits. Sub unit omega is needed for interaction with ppGpp, a regulatory molecule in stringent control.5) Novel modified forms of RNA polymerase holoenzyme exist in stationary-phase E. coli cells, each recognizing a different set of promoters.These results altogether indicate that global control of gene transcription takes place mainly by modulating the functions of RNA polymerase.