Multiplexed Proteomics Mapping of Yeast RNA Polymerase II and III Allows Near-Complete Sequence Coverage and Reveals Several Novel Phosphorylation Sites

The multisubunit RNA polymerases (Pols) II and III synthesize mainly eukaryotic mRNAs and tRNAs, respectively. Pol II and Pol III are protein complexes consisting of 12 and 17 subunits. Here we analyzed both yeast Pol II and Pol III by multiplexed mass spectrometric analysis using various proteases and both collision induced and electron transfer dissociation. The cumulative data obtained from using the various proteases (trypsin, chymotrypsin, Glu-C and Lys-C) and the two peptide fragmentation approaches allowed us to map nearly the complete sequences of all constituents of both Pol II and III. Notably, chymotrypsin behaved equally well as and in certain circumstances better than trypsin in the context of protein coverage. Although the available high resolution structures have exposed extensive mechanistic insights into transcription, the role of post-translational modification in these processes has been addressed to a lesser extent. In our analysis of Pol II and III we detected 19 phosphorylation sites, of which 12 have not been previously reported. Identified phosphosites were mapped on the Pol II structure which provided indications that they might play a role in regulating the conformation of the clamp region and, as a consequence, interaction of Pol II with nucleic acids. The described multiplexed proteomics approach is generic and reveals that it is possible to map a protein complex to near completion while applying less than 5 μg (approximately 10 pmol) of total starting material.

多亚基RNA聚合酶（multisubunit RNA polymerases，Pols）II与III分别主要负责合成真核生物的mRNA与tRNA。Pol II与Pol III均为蛋白质复合物，分别由12个与17个亚基构成。本研究通过多种蛋白酶结合碰撞诱导解离（collision induced dissociation, CID）与电子转移解离（electron transfer dissociation, ETD）的多重质谱分析方法，对酵母来源的Pol II与Pol III进行了分析。通过使用胰蛋白酶（trypsin）、胰凝乳蛋白酶（chymotrypsin）、Glu-C与Lys-C等多种蛋白酶，以及上述两种肽段碎裂策略所获得的累积数据，使得我们得以绘制出Pol II与Pol III所有组成成分的近乎完整序列图谱。值得注意的是，在蛋白质序列覆盖度层面，胰凝乳蛋白酶的表现与胰蛋白酶不相上下，在特定实验条件下甚至更胜一筹。尽管现有的高分辨率结构已为转录过程的机制研究提供了大量深入见解，但翻译后修饰（post-translational modification）在该过程中的作用却较少得到探讨。在对Pol II与Pol III的分析中，我们共检测到19个磷酸化位点，其中12个为此前未见报道的新位点。所鉴定的磷酸化位点被定位至Pol II的三维结构上，结果提示这些位点可能参与调控钳状结构域（clamp region）的构象，进而影响Pol II与核酸的相互作用。本研究所述的多重蛋白质组学方法具有普适性，研究表明仅需使用少于5 μg（约10 pmol）的总起始材料，即可实现对蛋白质复合物近乎完整的序列图谱绘制。