Department of Chemistry, Chemistry Research Laboratory, Mansfield Road, Oxford OX1 3TA, UK.
Structure. 2011 Jan 12;19(1):90-100. doi: 10.1016/j.str.2010.11.009.
RNA polymerases are essential enzymes which transcribe DNA into RNA. Here, we obtain mass spectra of the cellular forms of apo and holo eukaryotic RNA polymerase I and III, defining their composition under different solution conditions. By recombinant expression of subunits within the initiation heterotrimer of Pol III, we derive an interaction network and couple this data with ion mobility data to define topological restraints. Our data agree with available structural information and homology modeling and are generally consistent with yeast two hybrid data. Unexpectedly, elongation complexes of both Pol I and III destabilize the assemblies compared with their apo counterparts. Increasing the pH and ionic strength of apo and holo forms of Pol I and Pol III leads to formation of at least ten stable subcomplexes for both enzymes. Uniquely for Pol III many subcomplexes contain only one of the two largest catalytic subunits. We speculate that these stable subcomplexes represent putative intermediates in assembly pathways.
RNA 聚合酶是将 DNA 转录为 RNA 的必需酶。在这里，我们获得了无辅基和全辅基真核 RNA 聚合酶 I 和 III 的细胞形式的质谱，定义了它们在不同溶液条件下的组成。通过 Pol III 起始异三聚体的重组表达，我们得出了相互作用网络，并将该数据与离子淌度数据相结合，以定义拓扑限制。我们的数据与现有的结构信息和同源建模一致，并且通常与酵母双杂交数据一致。出乎意料的是，与无辅基形式相比，Pol I 和 III 的延伸复合物会使组装体不稳定。增加 apo 和 holo 形式的 Pol I 和 Pol III 的 pH 值和离子强度会导致两种酶形成至少十个稳定的亚基。对于 Pol III 来说，独特的是，许多亚基只包含两个最大的催化亚基之一。我们推测这些稳定的亚基代表组装途径中的潜在中间产物。
