Gonzalez B, Vasquez C, Bull P
DNA. 1984 Jun;3(3):251-7. doi: 10.1089/dna.1.1984.3.251.
The binding sites of Escherichia coli RNA polymerase to plasmid DNA from extremely thermophilic bacteria have been mapped by electron microscopy. Templates used in these studies included plasmids pTF62 (from Thermus flavus AT62) and pTT8 (from T. thermophilus HB8) and also hybrid molecules constructed by ligation of these plasmids to pBR322. Although the affinity of the enzyme for heterologous DNA was about one-third of that for pBR322, it was possible to localize preferred binding sites on pTF62 and pTT8. Six binding sites were identified in pTT8, mapping close to 7, 28, 47, 61, 65, and 81 map units (one unit being equal to 1% of the length of the DNA). Seven such regions located at 3, 27, 48, 60, 67, 81, and 86 map units were found in pTF62. RNA polymerase binding sites found in pBR322 coincided with promoters identified previously by electron microscopy analysis of transcriptional complexes prepared in vitro. These data indicate that E. coli RNA polymerase binds preferentially to specific sequences in plasmids from thermophilic bacteria, suggesting possible promoter locations in these plasmids.
通过电子显微镜已绘制出大肠杆菌RNA聚合酶与嗜热细菌质粒DNA的结合位点。这些研究中使用的模板包括质粒pTF62（来自嗜热栖热菌AT62）和pTT8（来自嗜热栖热菌HB8），以及通过将这些质粒与pBR322连接构建的杂交分子。尽管该酶对异源DNA的亲和力约为对pBR322亲和力的三分之一，但仍有可能在pTF62和pTT8上定位优先结合位点。在pTT8中鉴定出六个结合位点，位于靠近7、28、47、61、65和81个图距单位处（一个单位等于DNA长度的1%）。在pTF62中发现了七个位于3、27、48、60、67、81和86个图距单位处的此类区域。在pBR322中发现的RNA聚合酶结合位点与先前通过对体外制备的转录复合物进行电子显微镜分析鉴定的启动子一致。这些数据表明大肠杆菌RNA聚合酶优先结合嗜热细菌质粒中的特定序列，提示这些质粒中可能的启动子位置。
