Lorimer D D, Cao J L, Revzin A
Department of Biochemistry, Michigan State University, East Lansing 48824-1319.
J Mol Biol. 1990 Nov 20;216(2):275-87. doi: 10.1016/s0022-2836(05)80319-5.
A series of deletion mutants of the wild-type Escherichia coli lactose promoter, with endpoints at +25, +19, +14, +1 and -6 (relative to the start of transcription at +1), was constructed and the deleted DNA replaced with non-lac DNA. These mutants were used to show that no specific DNA sequences downstream from -6 are required for efficient promoter utilization in vitro. In all cases transcription is dependent on the presence of the catabolite activator protein (CAP) and cAMP, and begins at +1 at a level indistinguishable from that at the wild-type promoter. A set of lac DNA fragments deleted to -6 was constructed, having an A, C, G or T residue at +1 and heterologous DNA downstream. These synthetic promoters allow systematic testing of the effect of the initiating nucleotide on the transcription process. Again, transcription occurs mainly from +1, at a level similar to the normal wild-type level. No substantial differences between these promoters are observed in the rates of formation of stable complexes, in the degree of complex formation, in the rate at which polymerase "escapes" from the complex or in abortive transcription products. Equivalent results are seen with a related set of constructs based on the CAP-insensitive lac UV5 promoter. Thus, lac promoter sequences including consensus hexamers at -10 and -35, plus the spacer region between them, provide specificity and efficiency both in initiation of transcription by RNA polymerase and in CAP-polymerase interactions. A question as to whether there is a third RNA polymerase binding site at lac, in addition to the known overlapping P1 and P2 regions, was not unambiguously answered. However, if a "P3" site does exist, it must lie between P1 and P2. Alternatively, the variety of polymerase interactions at wild-type lac may reflect different structural states of the enzyme. The results presented here indicate that DNA downstream from -6 plays little part in determining the conformation of the enzyme at the lactose promoter.
构建了一系列野生型大肠杆菌乳糖启动子的缺失突变体，其端点分别位于 +25、+19、+14、+1 和 -6（相对于转录起始点 +1），并将缺失的 DNA 替换为非乳糖操纵子 DNA。这些突变体用于表明，在体外高效利用启动子时，-6 下游不需要特定的 DNA 序列。在所有情况下，转录都依赖于分解代谢物激活蛋白（CAP）和 cAMP 的存在，并且从 +1 开始，其水平与野生型启动子的水平无法区分。构建了一组缺失至 -6 的乳糖操纵子 DNA 片段，在 +1 处具有 A、C、G 或 T 残基，下游为异源 DNA。这些合成启动子允许系统地测试起始核苷酸对转录过程的影响。同样，转录主要从 +1 开始，水平与正常野生型水平相似。在稳定复合物形成速率、复合物形成程度、聚合酶从复合物中“逃逸”的速率或流产转录产物方面，未观察到这些启动子之间存在实质性差异。基于 CAP 不敏感的乳糖 UV5 启动子的相关构建体组也得到了相同的结果。因此，包括 -10 和 -35 处的共有六聚体以及它们之间的间隔区的乳糖启动子序列，在 RNA 聚合酶启动转录以及 CAP - 聚合酶相互作用方面都提供了特异性和效率。除了已知的重叠 P1 和 P2 区域外，乳糖操纵子是否存在第三个 RNA 聚合酶结合位点的问题尚未得到明确解答。然而，如果“P3”位点确实存在，它必定位于 P1 和 P2 之间。或者，野生型乳糖操纵子处多种聚合酶相互作用可能反映了酶的不同结构状态。此处呈现的结果表明，-6 下游的 DNA 在确定乳糖启动子处酶的构象方面作用很小。
