Pavco P A, Steege D A
Department of Biochemistry, Duke University Medical Center, Durham, NC 27710.
Nucleic Acids Res. 1991 Sep 11;19(17):4639-46. doi: 10.1093/nar/19.17.4639.
As a means of generating homogeneous populations of elongation complexes with the RNA polymerases encoded by phages T7 and SP6, transcription has been carried out in vitro on templates associated with the Gln-111 mutant of EcoRI endonuclease. The Gln-111 protein, as a result of a single amino acid substitution at position 111, lacks cleavage function yet shows higher than wild-type affinity for the EcoRI recognition sequence GAATTC. On a series of linear and circular templates associated with Gln-111 protein, blockage of the phage RNA polymerase elongation complex is observed. The 3' endpoint of the major blocked-length RNA species, just 3 bp upstream from the GAATTC, reveals an extremely close approach of polymerase's leading edge to essential contacts between Gln-111 protein and its binding site. In contrast to E. coli RNA polymerase, which is blocked stably and quantitatively by Gln-111 protein (Pavco, P.A. and Steege, D. A. (1990) J. Biol. Chem. 265, 9960-9969), the phage polymerases show substantial levels of readthrough transcription beyond the protein block.
作为一种生成由噬菌体T7和SP6编码的RNA聚合酶的均一延伸复合物群体的方法，已在与EcoRI核酸内切酶的Gln - 111突变体相关的模板上进行了体外转录。由于在第111位的单个氨基酸取代，Gln - 111蛋白缺乏切割功能，但对EcoRI识别序列GAATTC表现出高于野生型的亲和力。在与Gln - 111蛋白相关的一系列线性和环状模板上，观察到噬菌体RNA聚合酶延伸复合物的阻滞。主要阻滞长度RNA物种的3'末端，恰好在GAATTC上游3个碱基对处，揭示了聚合酶前沿与Gln - 111蛋白与其结合位点之间的关键接触极其接近。与被Gln - 111蛋白稳定且定量阻滞的大肠杆菌RNA聚合酶相反（Pavco, P.A.和Steege, D.A.（1990）J. Biol. Chem. 265, 9960 - 9969），噬菌体聚合酶在蛋白阻滞位点之后表现出相当水平的通读转录。
