Swanson M E, Yip M, Holland M J
J Biol Chem. 1985 Aug 15;260(17):9905-15.
Nucleotide sequences which are required for RNA polymerase I-dependent selective initiation of transcription in vitro from a site within the spacer region of cloned yeast ribosomal DNA have been identified. Yeast rDNA templates containing deletion mutations extending from restriction endonuclease cleavage sites located upstream and downstream from the transcriptional initiation site were constructed. The ability of these mutant templates to support selective transcription in vitro was determined using a yeast whole cell extract. Nucleotide sequences which are required for selective transcription in vitro are within a 22-base pair region which is located immediately adjacent to the transcriptional initiation site. The 3' boundary of this 22-base pair sequence was mapped within a single base pair and resides within the transcribed portion of the rDNA. Nucleotide sequences upstream and downstream from the 22-base pair region are not required for selective transcription and do not appear to affect the efficiency of transcription in vitro. A hybrid plasmid containing only 32 base pairs of yeast rDNA, which includes the 22-base pair region, supports efficient and accurate RNA polymerase I-dependent transcription in vitro. These data demonstrate that the 22-base pair region of yeast rDNA is sufficient for accurate initiation of transcription in vitro. The transcriptional properties of several cloned rDNA templates isolated from two haploid yeast strains and a strain of bakers' yeast were examined. Four cistrons were identified which differ in nucleotide sequence. Three cistrons contain the 22-base pair promoter region and they support selective transcription in vitro. The fourth cistron does not support selective transcription in vitro and contains a single base pair substitution within the 22-base pair promoter sequence.
已鉴定出在体外从克隆的酵母核糖体DNA间隔区的一个位点进行RNA聚合酶I依赖性选择性转录起始所需的核苷酸序列。构建了含有从转录起始位点上游和下游的限制性内切酶切割位点延伸的缺失突变的酵母rDNA模板。使用酵母全细胞提取物测定这些突变模板在体外支持选择性转录的能力。体外选择性转录所需的核苷酸序列位于紧邻转录起始位点的一个22碱基对区域内。这个22碱基对序列的3'边界定位在单个碱基对内，位于rDNA的转录部分内。22碱基对区域上游和下游的核苷酸序列对于选择性转录不是必需的，并且似乎不影响体外转录效率。一个仅包含32个碱基对酵母rDNA（包括22碱基对区域）的杂交质粒在体外支持高效且准确的RNA聚合酶I依赖性转录。这些数据表明酵母rDNA的22碱基对区域足以在体外准确起始转录。检查了从两个单倍体酵母菌株和一个面包酵母菌株分离的几个克隆rDNA模板的转录特性。鉴定出四个在核苷酸序列上不同的顺反子。三个顺反子包含22碱基对启动子区域，它们在体外支持选择性转录。第四个顺反子在体外不支持选择性转录，并且在22碱基对启动子序列内含有一个单碱基对替换。
