Shanblatt S H, Revzin A
Biochemistry. 1986 Sep 23;25(19):5539-46. doi: 10.1021/bi00367a029.
The molecular mechanisms whereby RNA polymerase, catabolite activator protein (CAP), and cyclic AMP (cAMP) participate in transcriptional regulation at the galactose operon have been probed by a variety of in vitro techniques. Interactions between purified proteins and promoter-containing DNA fragments were assayed by gel electrophoresis, by resistance to restriction endonuclease digestion, and by monitoring runoff transcripts. The data bear on the multiple functions that CAP performs in gal control. A CAP-cAMP complex can exclude RNA polymerase from one of the two overlapping promoter regions (P2), thereby targeting the enzyme to the other (P1); this process is markedly influenced by the cAMP level. In addition, a second CAP molecule is involved in a cooperative process, which, at low cAMP, is required for efficient formation of transcriptionally competent complexes at P1. This second CAP may serve to stabilize the 1:1:1 CAP-polymerase-gal DNA intermediate under physiological conditions, thus enhancing initiation from P1 relative to P2. Kinetic analysis reveals that the modest effect of CAP on the rate of P1 open complex formation can be resolved into about a 4-fold increase in the binding of RNA polymerase to the P1 region, plus a 1.5-fold elevation in the rate of isomerization of enzyme-promoter complexes to the open state.
通过多种体外技术探究了RNA聚合酶、分解代谢物激活蛋白（CAP）和环磷酸腺苷（cAMP）参与半乳糖操纵子转录调控的分子机制。通过凝胶电泳、对限制性内切酶消化的抗性以及监测连续转录本，分析了纯化蛋白与含启动子的DNA片段之间的相互作用。这些数据揭示了CAP在半乳糖操纵子控制中发挥的多种功能。CAP-cAMP复合物可将RNA聚合酶从两个重叠启动子区域之一（P2）排除，从而将该酶导向另一个区域（P1）；这一过程受cAMP水平的显著影响。此外，第二个CAP分子参与一个协同过程，在低cAMP水平时，该过程是在P1处有效形成具有转录活性的复合物所必需的。这第二个CAP可能在生理条件下稳定1:1:1的CAP-聚合酶-半乳糖DNA中间体，从而相对于P2增强从P1起始的转录。动力学分析表明，CAP对P1开放复合物形成速率的适度影响可分解为RNA聚合酶与P1区域结合增加约4倍，加上酶-启动子复合物异构化为开放状态的速率提高1.5倍。
