The Streptomyces galP1 promoter has a novel RNA polymerase recognition sequence and is transcribed by a new form of RNA polymerase in vitro

We report the identification of DNA sequences that determine the activity of the Streptomyces galP1 promoter and a new form of RNA polymerase holoenzyme that recognizes these sequences in vitro. Base substitutions were introduced throughout the galP1 promoter region, and bases at positions -34, -36, and -11 with respect to the transcription start site were shown to be required for promoter function. These bases correspond in their positions to regions known to be important for RNA polymerase binding in several classes of eubacterial promoters, but the sequences themselves are not similar to those previously described. The -35 region of the galP1 promoter consists of six G residues, and base changes in this G hexamer had a dramatic effect on promoter activity. By using galP1-containing DNA template, a new RNA polymerase activity was purified from Streptomyces. Holoenzyme reconstitution experiments identified a new sigma factor that directs galP1 transcription in vitro. DNase I protection experiments identified a binding site for this new holoenzyme immediately upstream of the galP1 transcription start site.

我们报道了对决定链霉菌galP1启动子活性的DNA序列的鉴定，以及一种在体外能识别这些序列的新型RNA聚合酶全酶。在galP1启动子区域各处引入了碱基替换，并且相对于转录起始位点在-34、-36和-11位置的碱基被证明是启动子功能所必需的。这些碱基在其位置上对应于已知在几类真细菌启动子中对RNA聚合酶结合很重要的区域，但这些序列本身与先前描述的那些并不相似。galP1启动子的-35区域由6个G残基组成，并且这个G六聚体中的碱基变化对启动子活性有显著影响。通过使用含galP1的DNA模板，从链霉菌中纯化出一种新的RNA聚合酶活性。全酶重组实验鉴定出一种新的σ因子，它在体外指导galP1转录。DNA酶I保护实验确定了这种新全酶在galP1转录起始位点紧邻上游的一个结合位点。