Mechanism of transcription of ribosomal DNA in vitro

1. SpRPA 12亚基是裂殖酵母核糖体DNA（rDNA）在体外高效转录所必需的。(1)SpRPA 1 2亚基的体外功能分析从野生型和Sprpa 12破坏菌株制备S100级分，以比较突变体相对于野生型的rDNA转录活性。使用各自的提取物进行体外转录，并通过Si核酸酶图谱检测体外合成的rRNA。结果表明，与野生型相比，Sprpa 12突变体提取物仅保留10%的转录活性，表明SpRPA 12亚基是在裂殖酵母中有效转录rDNA所必需的。还检查了各个提取物的转录活性的热不稳定性，表明SpRPA 12是RNA聚合酶I或假定的预起始复合物的热稳定性所需的。(2)SpRPA 12的体内功能分析。对SpRPA 12的C-末端区域进行缺失分析，结果表明在RPA 12同源物中保守的C-末端锌指基序对于SpRPA 12的功能不是必需的。含N端锌指基序的N端区域的缺失分析正在进行中. RPA 42亚基的表征。我们分离并鉴定了裂殖酵母Pol I的RPA 42亚基。结果表明，RPA 42与RPA 17亚基特异性相互作用，二者与原核生物RNA聚合酶α亚基具有同源性，提示RPA 42和RPA 17构成了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.