Ikeda R A
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta 30332-0400.
J Biol Chem. 1992 Jun 5;267(16):11322-8.
Promoter strength has been defined as the relative production of transcripts from a promoter. For T7 transcription it has frequently been observed that T7 class III promoters are qualitatively stronger than T7 class II promoters. In previous work it was observed that the maximum rates of initiation of three class III and three class II promoters show no class distinctions (Ikeda, R. A., Lin, A. C., and Clarke, J. (1992) J. Biol. Chem. 267, 2640-2649). This suggests that the efficiency of the conversion of the polymerase initiation complex to a stable transcription complex contributes to the overall strength of T7 promoters. The class differences in the strengths of T7 class II and class III promoters are confirmed by measuring the relative synthesis of run-off transcripts. These results show that the relative strengths of the class III promoters, phi 6.5, phi 10, and phi 13, are all comparable ranging from 0.61 for phi 6.5 to 1.00 for phi 10, while the relative strengths of the T7 class II promoters, phi 1.1B, phi 1.3, and phi 3.8, vary widely. One T7 class II promoter, phi 1.1B (relative strength = 0.34), approaches the strength of the class III promoters, while the other T7 class II promoters, phi 1.3 (relative strength = 0.045) and phi 3.8 (relative strength = 0.070) are nearly inactive. The efficiency of promoter clearance is then determined by measuring the relative production of small transcription products in comparison with the production of run-off transcripts. These measurements clearly distinguish the T7 class III promoters from the T7 class II promoters. It is found that 68-75% of all initiations at the T7 class III promoters phi 6.5, phi 10, and phi 13 produce a run-off transcript, while only 16-36% of the initiations at the T7 class II promoters phi 1.1B, phi 1.3, and phi 3.8 produce a run-off transcript. Clearly, promoter clearance contributes to the difference in promoter strengths of the T7 class II and III promoters.
启动子强度被定义为启动子转录本的相对产量。对于T7转录，人们经常观察到T7 III类启动子在性质上比T7 II类启动子更强。在之前的工作中，人们观察到三个III类启动子和三个II类启动子的最大起始速率没有类别差异（池田，R.A.，林，A.C.，和克拉克，J.（1992年）《生物化学杂志》267，2640 - 2649）。这表明聚合酶起始复合物转化为稳定转录复合物的效率有助于T7启动子的整体强度。通过测量连续转录本的相对合成量，证实了T7 II类和III类启动子强度的类别差异。这些结果表明，III类启动子phi 6.5、phi 10和phi 13的相对强度都相当，phi 6.5为0.61，phi 10为1.00，而T7 II类启动子phi 1.1B、phi 1.3和phi 3.8的相对强度差异很大。一个T7 II类启动子phi 1.1B（相对强度 = 0.34）接近III类启动子的强度，而其他T7 II类启动子phi 1.3（相对强度 = 0.045）和phi 3.8（相对强度 = 0.070）几乎没有活性。然后通过测量与连续转录本产量相比的小转录产物的相对产量来确定启动子清除效率。这些测量清楚地将T7 III类启动子与T7 II类启动子区分开来。发现在T7 III类启动子phi 6.5、phi 10和phi 13处，所有起始事件中有68 - 75%产生连续转录本，而在T7 II类启动子phi 1.1B、phi 1.3和phi 3.8处，只有16 - 36%的起始事件产生连续转录本。显然，启动子清除导致了T7 II类和III类启动子强度的差异。
