Osborne B I, Guarente L
Massachusetts Institute of Technology, Department of Biology, Cambridge 02139.
Genes Dev. 1988 Jun;2(6):766-72. doi: 10.1101/gad.2.6.766.
We show that induction of transcription of a CYC1-lacZ fusion gene, borne on a yeast plasmid, causes an increase in negative superhelicity of approximately five turns. This increase is abolished by deletion of either essential element of the CYC1 promoter, the upstream activation site (UAS), or the TATA boxes. Several experiments indicate that the size of the increase is proportional to the size of the transcribed region. First, an internal deletion removing half of the CYC1-lacZ transcribed region results in a plasmid whose negative superhelicity on induction is intermediate between promoter-deletion plasmids and the parental plasmid. Second, plasmids bearing insertions of a fragment containing the putative CYC1 terminator into the CYC1-lacZ fusion gene have relative negative superhelicities proportional to the length of the truncated fusion transcripts generated. A plausible model explaining these observations is that local unwinding of the double helix by transcribing RNA polymerase generates positively supercoiled DNA, which is subsequently relaxed by a topoisomerase.
我们发现，酵母质粒携带的CYC1-lacZ融合基因转录的诱导会导致负超螺旋增加约五圈。CYC1启动子的任何一个必需元件，即上游激活位点（UAS）或TATA框的缺失，都会消除这种增加。几个实验表明，增加的幅度与转录区域的大小成正比。首先，内部缺失CYC1-lacZ转录区域的一半会产生一种质粒，其诱导时的负超螺旋介于启动子缺失质粒和亲本质粒之间。其次，携带将包含推定的CYC1终止子的片段插入CYC1-lacZ融合基因的质粒，其相对负超螺旋与产生的截短融合转录本的长度成正比。一个解释这些观察结果的合理模型是，转录RNA聚合酶使双螺旋局部解旋产生正超螺旋DNA，随后由拓扑异构酶使其松弛。
