Westin G, Djurhuus R, Skreslett U
Biochim Biophys Acta. 1982 Oct 29;699(1):28-39. doi: 10.1016/0167-4781(82)90168-3.
RNA polymerase was purified from 'late' phage T4-infected Escherichia coli B cells by DNA-cellulose affinity chromatography and high salt agarose filtration. The DNA-cellulose-purified RNA polymerase preparation contained T4-coded DNA endonuclease activity and several proteins, some with sizes comparable with the known T4 maturation factors, essential for late RNA synthesis. Some of these proteins, and the DNA endonuclease utilizing native, parental T4 DNA and supercoiled phi X 174 DNA as substrates, were partially separated from the RNA polymerase as a complex during agarose filtration. In vitro RNA was made by the DNA-cellulose-purified RNA polymerase using native, parental T4 DNA as template. About 26% of the in vitro RNA was transcribed from the DNA r-strand; 75% from the same r-strand region as in vivo late after infection. Both the abundancy and specificity of the in vitro r-strand transcription were markedly reduced after agarose filtration of the enzyme. Addition of the proteins separated from the RNA polymerase during agarose filtration caused a restoration of in vitro r-strand transcription abundance, but not its specificity. These results show that partially purified RNA polymerase from T4-infected E. coli B cells was able to transcribe late T4 genes in vitro with some abundancy and specificity on purified, parental T4 DNA, but further purification of the enzyme caused an irreversible reduction of this ability.
通过DNA - 纤维素亲和层析和高盐琼脂糖过滤，从“晚期”噬菌体T4感染的大肠杆菌B细胞中纯化了RNA聚合酶。经DNA - 纤维素纯化的RNA聚合酶制剂含有T4编码的DNA内切酶活性和几种蛋白质，其中一些蛋白质的大小与已知的T4成熟因子相当，这些因子对晚期RNA合成至关重要。在琼脂糖过滤过程中，这些蛋白质中的一些以及以天然的亲本T4 DNA和超螺旋φX 174 DNA为底物的DNA内切酶作为一个复合物与RNA聚合酶部分分离。使用天然的亲本T4 DNA作为模板，由经DNA - 纤维素纯化的RNA聚合酶在体外合成RNA。约26%的体外RNA是从DNA的r链转录而来；75%是从感染后体内晚期相同的r链区域转录而来。在对该酶进行琼脂糖过滤后，体外r链转录的丰度和特异性均显著降低。添加在琼脂糖过滤过程中从RNA聚合酶中分离出的蛋白质可使体外r链转录丰度恢复，但不能恢复其特异性。这些结果表明，从T4感染的大肠杆菌B细胞中部分纯化的RNA聚合酶能够在体外以一定的丰度和特异性在纯化的亲本T4 DNA上转录晚期T4基因，但该酶的进一步纯化导致这种能力不可逆地降低。
