Differential Phosphorylation of RNA Polymerase III and the Initiation Factor TFIIIB in Saccharomyces cerevisiae

The production of ribosomes and tRNAs for protein synthesis has a high energetic cost and is under tight transcriptional control to ensure that the level of RNA synthesis is balanced with nutrient availability and the prevailing environmental conditions. In the RNA polymerase (pol) III system in yeast, nutrients and stress affect transcription through a bifurcated signaling pathway in which protein kinase A (PKA) and TORC1 activity directly or indirectly, through downstream kinases, alter the phosphorylation state and function of the Maf1 repressor and Rpc53, a TFIIF-like subunit of the polymerase. However, numerous lines of evidence suggest greater complexity in the regulatory network including the phosphoregulation of other pol III components. To address this issue, we systematically examined all 17 subunits of pol III along with the three subunits of the initiation factor TFIIIB for evidence of differential phosphorylation in response to inhibition of TORC1. A relatively high stoichiometry of phosphorylation was observed for several of these proteins and the Rpc82 subunit of the polymerase and the Bdp1 subunit of TFIIIB were found to be differentially phosphorylated. Bdp1 is phosphorylated on four major sites during exponential growth and the protein is variably dephosphorylated under conditions that inhibit tRNA gene transcription. PKA, the TORC1-regulated kinase Sch9 and protein kinase CK2 are all implicated in the phosphorylation of Bdp1. Alanine substitutions at the four phosphosites cause hyper-repression of transcription indicating that phosphorylation of Bdp1 opposes Maf1-mediated repression. The new findings suggest an integrated regulatory model for signaling events controlling pol III transcription.用于蛋白质合成的核糖体与转运RNA（transfer RNA, tRNA）的合成具有极高的能量成本，且受到严格的转录调控，以确保RNA合成水平与营养可获得性及当前环境条件相平衡。在酵母的RNA聚合酶III（RNA polymerase (pol) III）系统中，营养与压力通过一条分叉信号通路影响转录：蛋白激酶A（protein kinase A, PKA）与TORC1的活性可通过下游激酶直接或间接改变阻遏蛋白Maf1以及该聚合酶的TFIIF样亚基Rpc53的磷酸化状态与功能。然而，多项研究证据表明该调控网络具有更高的复杂性，其中还涵盖聚合酶III其他组分的磷酸化调控。为解决这一问题，我们系统检测了聚合酶III全部17个亚基以及起始因子TFIIIB的3个亚基，以探究其在TORC1抑制作用下的差异磷酸化特征。我们在多个靶蛋白中观察到了相对较高的磷酸化化学计量比，且发现该聚合酶的Rpc82亚基与TFIIIB的Bdp1亚基存在差异磷酸化现象。Bdp1在指数生长期会被磷酸化于四个主要位点，而在抑制tRNA基因转录的条件下，该蛋白会发生不同程度的去磷酸化。PKA、受TORC1调控的激酶Sch9以及蛋白激酶CK2均参与了Bdp1的磷酸化过程。对四个磷酸化位点进行丙氨酸替换会导致转录过度阻遏，这表明Bdp1的磷酸化可拮抗Maf1介导的转录阻遏作用。本研究的新发现为调控聚合酶III转录的信号事件提供了一套整合性的调控模型。