Analysis of the Functional Domains of RNA Polymerase

大肠杆菌的dna依赖性RNA聚合酶由α、β、β′和sigma四个主要亚基组成，不仅具有RNA合成的催化活性，还具有遗传信息传递的复杂功能。为了了解这些多重功能的结构基础，我们对大肠杆菌RNA聚合酶各亚基的功能域进行了系统的分析，得到了以下结果：1) α亚基在β和β′两个大亚基组装成酶复合物的过程中起着至关重要的作用。亚基组装需要n端区域，而位置因子结合启动子-35信号上游DNA的转录激活需要c端区域。突变RNA聚合酶由c端截断的α亚基组成，然而，被与启动子-35区域重叠的靶位点结合的阳性因子激活。2)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sigma结合位点。3)假设新的西格玛亚基已经确定，使用抗体针对合成肽与一致的氨基酸序列共同的各种西格玛亚基。编码这些蛋白质的基因正在使用基于这些氨基酸序列设计的DNA探针进行克隆。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。亚单位omega需要与ppGpp相互作用，ppGpp是一种严格控制的调节分子。5)在静止期大肠杆菌细胞中存在新的修饰形式的RNA聚合酶全酶，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是通过调节RNA聚合酶的功能来实现的。

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.