Stahl S J, Chamberlin M J
J Biol Chem. 1978 Jul 25;253(14):4951-9.
The interaction of bacteriophage T7 specific RNA polymerase with its cognate promoter sites has been probed by selectively replacing bases in one T7 promoter site with base analogs. Base analogs such as 2,6-diaminopurine or hypoxanthine, which alter residues appearing in the minor groove of the DNA helix, prevent utilization of the promoter by T7 RNA polymerase. These analogs do not affect transcription which starts outside of the modified region. In contrast, base analogs that have alterations that appear in the major groove of the DNA helix, such as uracil, 5-bromouracil, 5-methylcytosine, 5-hydroxymethylcytosine, and [5-HgSR]pyrimidines, do not prevent utilization of the promoter. The deoxyribonucleoside analog 5'-imino-5'-deoxythymidine, an alteration appearing in the deoxyribose-phosphodiester backbone of the DNA helix, does not prevent promoter recognition. Haemophilus aegyptius restriction endonuclease III, which cleaves DNA at the sequence 5'GGCC3', does not act at sites in which the guanine residues in one of the two DNA strands have been substituted with hypoxanthine. This implicates the guanine amino group in the minor groove of the DNA helix as a possible recognition point for this restriction endonuclease.
通过用碱基类似物选择性取代T7启动子位点中的碱基，对噬菌体T7特异性RNA聚合酶与其同源启动子位点之间的相互作用进行了研究。诸如2,6-二氨基嘌呤或次黄嘌呤等碱基类似物会改变出现在DNA螺旋小沟中的残基，从而阻止T7 RNA聚合酶利用该启动子。这些类似物不会影响在修饰区域之外起始的转录。相比之下，在DNA螺旋大沟中出现改变的碱基类似物，如尿嘧啶、5-溴尿嘧啶、5-甲基胞嘧啶、5-羟甲基胞嘧啶和[5-HgSR]嘧啶，不会阻止启动子的利用。脱氧核糖核苷类似物5'-亚氨基-5'-脱氧胸苷是出现在DNA螺旋脱氧核糖-磷酸二酯主链中的一种改变，它不会阻止启动子识别。埃及嗜血杆菌限制性内切酶III在5'GGCC3'序列处切割DNA，但不会作用于两条DNA链之一中的鸟嘌呤残基被次黄嘌呤取代的位点。这表明DNA螺旋小沟中的鸟嘌呤氨基可能是这种限制性内切酶的一个识别点。
