Brun I, Sentenac A, Werner M
Service de Biochimie et Génétique Moléculaire, Bâtiment 142, CEA/Saclay, F-91191 Gif-sur-Yvette Cedex, France.
EMBO J. 1997 Sep 15;16(18):5730-41. doi: 10.1093/emboj/16.18.5730.
The C34 subunit of yeast RNA polymerase (pol) III is part of a subcomplex of three subunits which have no counterpart in the other two nuclear RNA polymerases. This subunit interacts with TFIIIB70 and is therefore thought to participate in pol III recruitment. To study the role of C34 in transcription, we have mutagenized RPC34, the gene encoding C34, and found that mutations affecting growth also altered C34 interaction with TFIIIB70. The two mutant pol III that were purified had catalytic properties indistinguishable from those of the wild-type pol III on a poly[d(A-T)] template, while specific transcription of pol III genes in the presence of general transcription factors was impaired. The defect of the C34-1124 mutant enzyme could be compensated by increasing the amount of pol III present in the reaction, suggesting that the enzyme had a lower affinity for pre-initiation complexes. In contrast, the C34-1109 mutant enzyme was defective in transcription initiation due to impaired open complex formation. These observations demonstrate that the C34 subunit is a major determinant in pol III recruitment by the pre-initiation complex and further acts at a subsequent stage that involves the configuration of an initiation-competent form of RNA polymerase.
酵母RNA聚合酶（pol）III的C34亚基是一个由三个亚基组成的亚复合物的一部分，这三个亚基在其他两种核RNA聚合酶中没有对应物。该亚基与TFIIIB70相互作用，因此被认为参与了pol III的募集。为了研究C34在转录中的作用，我们对编码C34的基因RPC34进行了诱变，发现影响生长的突变也改变了C34与TFIIIB70的相互作用。纯化得到的两种突变型pol III在聚[d（A-T）]模板上的催化特性与野生型pol III无法区分，而在存在通用转录因子的情况下，pol III基因的特异性转录受到损害。C34-1124突变酶的缺陷可以通过增加反应中pol III的量来补偿，这表明该酶对预起始复合物的亲和力较低。相比之下，C34-1109突变酶由于开放复合物形成受损而在转录起始方面存在缺陷。这些观察结果表明，C34亚基是预起始复合物募集pol III的主要决定因素，并在随后涉及RNA聚合酶起始活性形式构象的阶段进一步发挥作用。
