Burrows Patricia C, Joly Nicolas, Nixon B Tracy, Buck Martin
Department of Life Sciences, Division of Biology, Faculty of Natural Sciences, Sir Alexander Fleming Building, Imperial College London, London SW7 2AZ, UK.
Nucleic Acids Res. 2009 Aug;37(15):5138-50. doi: 10.1093/nar/gkp541. Epub 2009 Jun 24.
Bacterial RNA polymerase (RNAP) containing the major variant sigma(54) factor forms open promoter complexes in a reaction in which specialized activator proteins hydrolyse ATP. Here we probe binding interactions between sigma(54)-RNAP (Esigma(54)) and the ATPases associated with various cellular activities (AAA+) domain of the Escherichia coli activator protein, PspF, using nucleotide-metal fluoride (BeF and AlF) analogues representing ground and transition states of ATP, which allow complexes (that are otherwise too transient with ATP) to be captured. We show that the organization and functionality of the ADP-BeF- and ADP-AlF-dependent complexes greatly overlap. Our data support an activation pathway in which the initial ATP-dependent binding of the activator to the Esigma(54) closed complex results in the re-organization of Esigma(54) with respect to the transcription start-site. However, the nucleotide-dependent binding interactions between the activator and the Esigma(54) closed complex are in themselves insufficient for forming open promoter complexes when linear double-stranded DNA is present in the initial closed complex.
含有主要变体σ⁵⁴因子的细菌RNA聚合酶（RNAP）在一种反应中形成开放启动子复合物，在该反应中，特殊的激活蛋白水解ATP。在这里，我们使用代表ATP基态和过渡态的核苷酸-金属氟化物（BeF和AlF）类似物，探测σ⁵⁴-RNAP（Eσ⁵⁴）与大肠杆菌激活蛋白PspF的各种细胞活动相关的ATP酶（AAA⁺）结构域之间的结合相互作用，这使得复合物（否则与ATP形成的复合物过于短暂）能够被捕获。我们表明，ADP-BeF-和ADP-AlF依赖性复合物的组织和功能有很大重叠。我们的数据支持一种激活途径，即激活剂最初依赖ATP与Eσ⁵⁴封闭复合物的结合导致Eσ⁵⁴相对于转录起始位点的重新组织。然而，当线性双链DNA存在于初始封闭复合物中时，激活剂与Eσ⁵⁴封闭复合物之间的核苷酸依赖性结合相互作用本身不足以形成开放启动子复合物。
