Two RNA polymerase I subunits control the binding and release of Rrn3 during transcription
Two RNA polymerase I subunits control the binding and release of Rrn3 during transcription
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DOI：
10.1128/mcb.01464-07
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发表时间：
2008-03-01
影响因子：
5.3
通讯作者：
Thuriaux, Pierre
中科院分区：
文献类型：
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作者：
Beckouet, Frederic;Labarre-Mariotte, Sylvie;Thuriaux, Pierre
Rpa34和Rpa49是RNA聚合酶1的非必需亚基，在酿酒酵母和裂殖酵母等物种中对人类具有保守性。在双杂交实验中，Rpa34与Rpa49的N-末端区域结合，并在缺少Rpa34结合结构域的rpa49突变体中从RNA聚合酶中丢失，而rpa34 Delta削弱了Rpa49与RNA聚合酶的结合。Rpa34 Delta突变体对咖啡因敏感，在top1 Delta突变体和rpa14 Delta、rpa135(L656P)和rpa135(D395N)RNA聚合酶突变体中，rpa34 Delta突变是致死的。这些缺陷被rpa49 Delta突变体所共有，并被Rpa49的过表达抑制，因此，推测是由Rpa49本身介导的。缺乏Rpa34结合结构域的rpa49突变体的行为基本上与rpa34 Delta突变体相似，但携带rpa49 Delta和rpa49-338：：HIS3的菌株(编码一种缺乏保守C末端的Rpa49)在30℃时聚合酶占有率降低，在25℃下不能生长，并且对6-氮卓菌素和霉酚酸酯敏感。霉酚酸酯几乎完全将突变的聚合酶与其核糖体DNA(RDNA)模板解离。Rpa49 Delta和rpa49-338：HIS3突变对转录起始因子Rrn3(TIF-IA)具有双重作用。它们部分地削弱了它对rDNA启动子的招募，这一影响被由rpa43-35,326编码的Rpa43亚基的N端缺失所绕过，并且它们强烈地减少了Rrn3启动因子在伸长过程中的释放。这些数据表明，Rpa49-Rpa34二聚体在Rrn3的招募和随后从延伸的聚合酶解离过程中具有双重作用。
Rpa34 and Rpa49 are nonessential subunits of RNA polymerase 1, conserved in species from Saccharomyces cerevisiae and Schizosaccharomyces pombe to humans. Rpa34 bound an N-terminal region of Rpa49 in a two-hybrid assay and was lost from RNA polymerase in an rpa49 mutant lacking this Rpa34-binding domain, whereas rpa34 Delta weakened the binding of Rpa49 to RNA polymerase. rpa34 Delta mutants were caffeine sensitive, and the rpa34 Delta mutation was lethal in a top1 Delta mutant and in rpa14 Delta, rpa135(L656P), and rpa135(D395N) RNA polymerase mutants. These defects were shared by rpa49 Delta mutants, were suppressed by the overexpression of Rpa49, and thus, were presumably mediated by Rpa49 itself. rpa49 mutants lacking the Rpa34-binding domain behaved essentially like rpa34 Delta mutants, but strains carrying rpa49 Delta and rpa49-338::HIS3 (encoding a form of Rpa49 lacking the conserved C terminus) had reduced polymerase occupancy at 30 degrees C, failed to grow at 25 degrees C, and were sensitive to 6-azauracil and mycophenotate. Mycophenolate almost fully dissociated the mutant polymerase from its ribosomal DNA (rDNA) template. The rpa49 Delta and rpa49-338:HIS3 mutations had a dual effect on the transcription initiation factor Rrn3 (TIF-IA). They partially impaired its recruitment to the rDNA promoter, an effect that was bypassed by an N-terminal deletion of the Rpa43 subunit encoded by rpa43-35,326, and they strongly reduced the release of the Rrn3 initiation factor during elongation. These data suggest a dual role of the Rpa49-Rpa34 dimer during the recruitment of Rrn3 and its subsequent dissociation from the elongating polymerase.