Ikeda R A, Richardson C C
J Biol Chem. 1987 Mar 15;262(8):3800-8.
The association of nicked RNA polymerase of bacteriophage T7 (Ikeda, R. A., and Richardson, C. C. (1987) J. Biol. Chem. 262, 3790-3799) with the T7 phi 10 promoter has been examined by DNA cleavage protection. The phi 10 promoter consists of a 23-base pair consensus sequence that extends from -17 to +6 with respect to the site of the initiation of transcription (+1). Nicked T7 RNA polymerase alone protects 20 bases from -21 to -2 (+/- 1) base at each border. Initiation and synthesis of the trinucleotide r(GGG) expands and shifts the sequence protected by nicked T7 RNA polymerase. Twenty-five bases are protected from -17 to +8 (+/- 1). The polymerization of three additional ribonucleotides, synthesis of the hexamer r(GGGAGA), further expands the protected sequence. Twenty-seven bases are protected from -17-+10 (+/- 1). Finally, the synthesis of a pentadecaribonucleotide transcript, r(GGGAGACCACGG), leads to the formation of a transcription complex that protects 22 bases from -2-+20 (+/- 1). In comparison to the sequences protected by T7 RNA polymerase the sequences protected by the nicked enzyme are shortened at the 5' end and are translocated downstream much earlier during the initiation of transcription. It appears that a portion of the DNA contacts made at the amino terminus of T7 RNA polymerase are disrupted in the small fragment of nicked T7 RNA polymerase. The changes that are observed in the sequences protected by nicked T7 RNA polymerase are reflected in the physical characteristics of the DNA X enzyme complexes. The number of ion pairs formed by the r(GGG)-initiated complex of the nicked enzyme is reduced, and the association constant for the formation of the r(GGG)-initiated complex is decreased as compared to the intact T7 RNA polymerase.
通过DNA切割保护实验研究了噬菌体T7带切口的RNA聚合酶（池田，R.A.，和理查森，C.C.（1987年）《生物化学杂志》262卷，3790 - 3799页）与T7 φ10启动子的结合情况。φ10启动子由一个23碱基对的共有序列组成，相对于转录起始位点（+1），该序列从 - 17延伸至 +6。单独的带切口的T7 RNA聚合酶在每个边界可保护从 - 21到 - 2（±1）的20个碱基。三磷酸鸟苷（r(GGG)）的起始合成会扩展并移动带切口的T7 RNA聚合酶所保护的序列。从 - 17到 +8（±1）有25个碱基受到保护。再添加三个核糖核苷酸进行聚合，即合成六聚体r(GGGAGA)，会进一步扩展受保护序列。从 - 17到 +10（±1）有27个碱基受到保护。最后，合成十五聚核糖核苷酸转录本r(GGGAGACCACGG)会导致形成一个转录复合物，该复合物可保护从 - 2到 +20（±1）的22个碱基。与T7 RNA聚合酶所保护的序列相比，带切口酶所保护的序列在5'端缩短，并且在转录起始过程中更早地向下游移位。看来在T7 RNA聚合酶氨基末端形成的一部分DNA接触在带切口的T7 RNA聚合酶的小片段中被破坏了。在带切口的T7 RNA聚合酶所保护的序列中观察到的变化反映在DNA - 酶复合物的物理特性上。与完整的T7 RNA聚合酶相比，带切口酶的r(GGG)起始复合物形成的离子对数量减少，并且r(GGG)起始复合物形成的结合常数降低。
