Carnevali F, Caserta M, Di Mauro E
Nucleic Acids Res. 1982 May 25;10(10):3195-209. doi: 10.1093/nar/10.10.3195.
Clones of the yeast Tyl element and 2 microns plasmid have been selectively transcribed in vitro by partially or completely purified yeast RNA polymerase II. Electrophoretic analysis of whole and restricted ternary transcription complexes allows the localization of the in vitro actively transcribed regions of the analyzed genes. The DNA regions that actively promote in vitro transcription correspond to the nucleotide sequence that in the Tyl element encompasses the in vivo transcription initiation sites and that in the 2 micrometer plasmid encompasses the starting codons of two oppositely oriented potential protein coding frames. The transcription assay that we describe herein may be applied to analyze rapidly the in vitro transcription of clones genes, to localize transcription initiation sites on supercoiled templates and to evaluate the differential in vitro promoter strength in RNA polymerase II served genes. Data obtained with RNA polymerase II at two different stages of purification are presented in parallel. Studies with a completely purified enzyme should certainly be preferred although the use of a partially purified RNA polymerase II may be convenient and may reveal factors which affect specificity.
酵母Tyl元件和2微米质粒的克隆已被部分或完全纯化的酵母RNA聚合酶II在体外选择性转录。对完整和限制性三元转录复合物的电泳分析可以确定所分析基因在体外的活跃转录区域。在体外积极促进转录的DNA区域对应于Tyl元件中包含体内转录起始位点的核苷酸序列，以及2微米质粒中包含两个方向相反的潜在蛋白质编码框架起始密码子的核苷酸序列。我们在此描述的转录分析方法可用于快速分析克隆基因的体外转录，定位超螺旋模板上的转录起始位点，并评估RNA聚合酶II作用基因的体外启动子强度差异。同时展示了在两个不同纯化阶段使用RNA聚合酶II获得的数据。尽管使用部分纯化的RNA聚合酶II可能很方便，并且可能揭示影响特异性的因素，但使用完全纯化的酶进行研究当然更可取。
