Facilitated Recycling Protects Human RNA Polymerase III from Repression by Maf1 in Vitro
Facilitated Recycling Protects Human RNA Polymerase III from Repression by Maf1 in Vitro
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DOI：
10.1074/jbc.m807538200
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发表时间：
2008-12-26
影响因子：
4.8
通讯作者：
Willis, Ian M.
中科院分区：
文献类型：
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作者：
Cabart, Pavel;Lee, JaeHoon;Willis, Ian M.
酵母细胞在每个细胞周期以2-4个转录本/基因/秒的速度合成大约3-6百万tRNA分子。这种高转录率是通过RNA聚合酶（pol） III在起始因子TFIIIB的稳定dna结合复合物上进行多轮再起始来实现的。对酵母的研究表明，再起始率通过促进再循环而增加，这一过程涉及在相同的转录单元上重复重新装载聚合酶。然而，当营养变得有限或遇到应激条件时，RNA pol III转录通过保守的Maf1蛋白的作用迅速受到抑制。在这里，我们研究了人类RNA pol III体外系统中maf1介导的抑制和促进循环之间的关系。使用固定化模板转录测定，我们证明了便利的再循环是从酵母到人类的保守。我们评估了重组人Maf1在起始前复合物组装前后抑制不同转录步骤的能力。我们发现重组Maf1可以抑制TFIIIB和RNA pol III向固定化模板的募集。然而，结合在起始前复合物或延伸复合物上的RNA pol III不受Maf1的抑制，可以经历几轮起始。这表明重组Maf1不能抑制促进再循环。这些数据表明，对于依赖于maf1的RNA pol III转录的快速抑制，可能需要额外的生化步骤。
Yeast cells synthesize similar to 3-6 million molecules of tRNA every cell cycle at a rate of similar to 2-4 transcripts/gene/s. This high rate of transcription is achieved through many rounds of reinitiation by RNA polymerase (pol) III on stable DNA-bound complexes of the initiation factor TFIIIB. Studies in yeast have shown that the rate of reinitiation is increased by facilitated recycling, a process that involves the repeated reloading of the polymerase on the same transcription unit. However, when nutrients become limiting or stress conditions are encountered, RNA pol III transcription is rapidly repressed through the action of the conserved Maf1 protein. Here we examine the relationship between Maf1-mediated repression and facilitated recycling in a human RNA pol III in vitro system. Using an immobilized template transcription assay, we demonstrate that facilitated recycling is conserved from yeast to humans. We assessed the ability of recombinant human Maf1 to inhibit different steps in transcription before and after preinitiation complex assembly. We show that recombinant Maf1 can inhibit the recruitment of TFIIIB and RNA pol III to immobilized templates. However, RNA pol III bound to preinitiation complexes or in elongation complexes is protected from repression by Maf1 and can undergo several rounds of initiation. This indicates that recombinant Maf1 is unable to inhibit facilitated recycling. The data suggest that additional biochemical steps may be necessary for rapid Maf1-dependent repression of RNA pol III transcription.