Mencía M, Monsalve M, Rojo F, Salas M
Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, Madrid, Spain.
J Mol Biol. 1998 Jan 16;275(2):177-85. doi: 10.1006/jmbi.1997.1463.
Regulatory protein p4 of Bacillus subtilis phage phi 29 activates transcription from the viral late A3 promoter by interacting with the C-terminal domain (CTD) of the B. subtilis RNA polymerase alpha subunit, thereby stabilizing the holoenzyme at the promoter. Protein p4 does not interact with the Escherichia coli RNA polymerase and cannot activate transcription with this enzyme. We have constructed a chimerical alpha subunit containing the N-terminal domain of the E. coli alpha subunit and the CTD of the B. subtilis alpha subunit. Reconstitution of RNA polymerases containing this chimerical alpha subunit, the E. coli beta and beta' subunits, and the vegetative sigma factor from either E. coli (sigma 70) or B. subtilis (sigma A), generated hybrid enzymes that were responsive to protein p4 and efficiently supported activation at the A3 promoter. Protein p4 activated transcription with the chimerical enzymes through the same activation surface used with B. subtilis RNA polymerase. Therefore, the B. subtilis alpha-CTD allowed activation by p4 even when the rest of the RNA polymerase subunits belonged to E. coli, a distantly related bacterium. These results strongly suggest that protein p4 works essentially by serving as an anchor that stabilizes RNA polymerase at the promoter.
枯草芽孢杆菌噬菌体phi 29的调控蛋白p4通过与枯草芽孢杆菌RNA聚合酶α亚基的C末端结构域（CTD）相互作用，激活病毒晚期A3启动子的转录，从而将全酶稳定在启动子上。蛋白p4不与大肠杆菌RNA聚合酶相互作用，也不能用这种酶激活转录。我们构建了一种嵌合α亚基，它包含大肠杆菌α亚基的N末端结构域和枯草芽孢杆菌α亚基的CTD。含有这种嵌合α亚基、大肠杆菌β和β'亚基以及来自大肠杆菌（σ70）或枯草芽孢杆菌（σA）的营养型σ因子的RNA聚合酶的重组，产生了对蛋白p4有反应并能有效支持A3启动子激活的杂交酶。蛋白p4通过与枯草芽孢杆菌RNA聚合酶相同的激活表面，用嵌合酶激活转录。因此，即使RNA聚合酶的其他亚基属于远缘相关细菌大肠杆菌，枯草芽孢杆菌α-CTD也能实现p4介导的激活。这些结果有力地表明，蛋白p4主要通过作为一种将RNA聚合酶稳定在启动子上的锚定物来发挥作用。
