T7 RNA Polymerase Functions In Vitro without Clustering

Many nucleic acid polymerases function in clusters known as factories. We investigate whether the RNA polymerase (RNAP) of phage T7 also clusters when active. Using ‘pulldowns’ and fluorescence correlation spectroscopy we find that elongation complexes do not interact in vitro with a KdE. coli chromosome do not associate more frequently when transcribed by T7 RNAP. We conclude that if clustering does occur in vivo, it must be driven by weak interactions, or mediated by a phage-encoded protein.

诸多核酸聚合酶会以被称为"工厂"的簇状结构行使功能。本研究旨在探究T7噬菌体的RNA聚合酶（RNA polymerase, RNAP）在激活状态下是否同样会发生聚集。我们借助下拉实验（pulldowns）与荧光相关光谱法开展实验，结果显示体外环境中的延伸复合物（elongation complexes）并不会以解离常数Kd发生相互作用；当大肠杆菌（E. coli）染色体被T7 RNAP转录时，其相互关联的频率也未出现升高。据此我们得出结论：若该聚合酶在体内确实发生聚集，则其驱动因素必然为弱相互作用，或是由某一噬菌体编码蛋白所介导。