Mechanism of transcription of ribosomal DNA in vitro

1.SpRPA12亚基是核糖体DNA(RDNA)在裂解酵母中高效转录所必需的。(1)SpRPA1-2亚基体外功能分析。从野生型和Sprpa12突变株中提取S100组分，比较突变体和野生型的rDNA转录活性。用各自的提取物进行体外转录，并用硅核酸酶图谱检测体外合成的rRNA。结果表明，Sprpa12突变体提取物与野生型相比只保留了10%的转录活性，这表明SpRPA12亚基是rDNA在分裂酵母中高效转录所必需的。还研究了各自提取物的转录活性的热稳定性，表明SpRPA12是RNA聚合酶I或推测的预起始复合体的热稳定性所必需的。(2)SpRPA12的体内功能分析。对SpRPA12的C-末端区域进行了缺失分析，结果表明，SpRPA12的功能并不需要在RPA12同源物中保守的C-末端锌指基序。含有N-末端锌指基序的N-末端区域的缺失分析目前正在进行中。RPA42亚基的特性。我们分离并鉴定了裂解酵母Pol I的RPA42亚基。结果表明，RPA42与RPA17亚基特异地相互作用，两者与原核生物中的RNA聚合酶α亚基具有同源性，表明RPA42和RPA17是Pol I的核心。

1. SpRPA12 subunit is required for efficient transcription of ribosomal DNA (rDNA) in the fission yeast in vitro. (1) Analysis of function of SpRPA1 2 subunit in vitro. S100 fraction was prepared from the wild-type and Sprpa12 disruptant strains to compare the rDNA transcription activity of the mutant relative to wild-type. In vitro transcription was carried out using the respective extracts and the rRNA synthesized in vitro was detected by Si nuclease mapping. The results have shown that Sprpa12 mutant extract retained only 10% transcription activity compared to wild-type, indicating that SpRPA12 subunit is required for the efficient transcription of rDNA in the fission yeast. The thermolability of the transcription activity of the respective extracts was also examined, suggesting that SpRPA12 is required for the thermostability of the RNA polymerase I or the presumed preinitiation complex.. (2) Analysis of function of SpRPA12 in vivo. The deletion analysis of the C-terminal region of SpRPA12 was performed and the results have shown that the C-terminal zinc-finger motif that is conserved among RPA12 homologs is not required for the function of SpRPA12. The deletion analysis of the N-terminal region containing the N-terminal zinc-finger motif is now on progress.2. Characterization of RPA42 subunit. We have isolated and characterized RPA42 subunit of the fission yeast Pol I. The results have shown that RPA42 interacts specifically with RPA17 subunit and both of them show homologies to alpha subunit of RNA polymerase in prokaryotes, suggesting that RPA42 and RPA17 form a core of Pol I.