Hsieh T, Wang J C
Biochemistry. 1976 Dec 28;15(26):5776-83. doi: 10.1021/bi00671a014.
The cleavage sites in the early promoter region of coliphage T7 have been mapped for four restriction enzymes. They are, from the left end in base pairs, 1100 and 740 for Hinf; 680, 320, 530, 240, 77, and 67 for Hind II; 620 and 530 for Hpa II; 790 for Alu I. The nucleotide sequence between the Hind II site at 680 base pairs from the left end and the Hinf site at 740 base pairs from the left end has been determined, from which the start point of the promoter A3 is located at 720 base pairs from the left end. The start points of the other two major promoters A1 and A2 are deduced to be at 460 and 580 base pairs from the left end, respectively, from the chain lengths of the in vitro transcripts off the 1100 base-pairs long Hinf fragment. Similar to the sequences of a pL and pR promotors of phage lambda and a sequence in Simian Virus 40 used by Escherichia coli RNA polymerase as a promotor, the sequence of the A3 promotor of T7 also has a Hind II restriction site approximately 30 base pairs upstream to the start point of RNA synthesis. No such Hind II sites exist, however, for the A1 and A2 promoters. Experiments on the protection of some of the restriction sites on the 1100 base-pairs-long Hinf fragment by RNA polymerase binding support the electron microscopic observations of others that, in addition to the three sites A1, A2 and A3, there is at least a fourth site at which E. coli RNA polymerase can bind strongly. In addition to the Hind II site at 680 base pairs from the left end and the Hinf site at 740 base pairs from the left end, which are presumably protected by the binding of a single RNA polymerase at the A3 site, the Hind II site at 240 base pairs from the left end is also protected at a level of 5 polymerase molecules/DNA fragment. The possible existence of several minor promotor sites in the early promotor region, in addition to the three major promotor sites, is discussed.
已确定了四种限制酶在大肠杆菌噬菌体T7早期启动子区域的切割位点。从左端起按碱基对计算，Hinf的切割位点为1100和740；Hind II的切割位点为680、320、530、240、77和67；Hpa II的切割位点为620和530；Alu I的切割位点为790。已确定了从左端起680个碱基对处的Hind II位点与左端起740个碱基对处的Hinf位点之间的核苷酸序列，由此可知启动子A3的起始点位于左端起720个碱基对处。根据1100个碱基对长的Hinf片段体外转录本的链长推断，另外两个主要启动子A1和A2的起始点分别位于左端起460和580个碱基对处。与噬菌体λ的pL和pR启动子序列以及大肠杆菌RNA聚合酶用作启动子的猿猴病毒40中的一个序列相似，T7的A3启动子序列在RNA合成起始点上游约30个碱基对处也有一个Hind II限制位点。然而，A1和A2启动子没有这样的Hind II位点。关于RNA聚合酶结合对1100个碱基对长的Hinf片段上一些限制位点的保护实验，支持了其他人的电子显微镜观察结果，即除了A1、A2和A3这三个位点外，至少还有第四个位点，大肠杆菌RNA聚合酶可以在该位点强烈结合。除了左端起680个碱基对处的Hind II位点和左端起740个碱基对处的Hinf位点（推测它们因单个RNA聚合酶在A3位点的结合而受到保护）外，左端起240个碱基对处的Hind II位点在每DNA片段5个聚合酶分子的水平下也受到保护。文中还讨论了除三个主要启动子位点外，早期启动子区域可能存在几个次要启动子位点的情况。
