Lescure B, Williamson V, Sentenac A
Nucleic Acids Res. 1981 Jan 10;9(1):31-45. doi: 10.1093/nar/9.1.31.
Yeast RNA polymerase B catalyzes an efficient abortive initiation on double-stranded DNA templates using the appropriate combination of primer and substrate. The specificity of initiation was investigated using a recombinant plasmid (pJD14 DNA) containing the structural gene for yeast alcohol dehydrogenase I (ADHI). The combination of the dinucleotide UpA and UTP was 10 fold more efficient with pJD14 DNA than with the vector pBR322 DNA to direct the synthesis of the trinucleotide UpApU. Under these conditions, stable enzyme-DNA complexes were formed and could be retained on nitrocellulose filters. Using the UpA-primed system and a short pulse of RNA synthesis, transcription complexes were located on the yeast part of pJD14 DNA as evidenced by agarose gel electrophoresis. Southern hybridization of the pulsed RNA was restricted to a region, within the yeast DNA fragment, upstream to the initial region of the ADHI gene.
酵母RNA聚合酶B使用合适的引物和底物组合，在双链DNA模板上催化高效的流产起始。使用含有酵母乙醇脱氢酶I（ADHI）结构基因的重组质粒（pJD14 DNA）研究起始的特异性。二核苷酸UpA和UTP的组合对pJD14 DNA指导合成三核苷酸UpApU的效率比对载体pBR322 DNA高10倍。在这些条件下，形成了稳定的酶-DNA复合物，并且可以保留在硝酸纤维素滤膜上。使用UpA引发系统和短时间的RNA合成脉冲，通过琼脂糖凝胶电泳证明转录复合物位于pJD14 DNA的酵母部分。脉冲RNA的Southern杂交局限于酵母DNA片段内ADHI基因起始区域上游的一个区域。
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