Colbert T, Hahn S
Fred Hutchinson Cancer Research Center, Seattle, Washington 98104-2092.
Genes Dev. 1992 Oct;6(10):1940-9. doi: 10.1101/gad.6.10.1940.
A suppressor gene was identified, which in high copy number rescues a temperature-sensitive mutation in yeast TATA-binding protein (TBP). Suppression was allele specific because the suppressor did not rescue the temperature-sensitive phenotype of another TBP mutant. This suppressor gene encodes a 596-amino-acid protein of which the amino-terminal half is homologous to the Pol II-specific factor TFIIB. Disruption of this gene, termed BRF1, showed it to be essential for growth of yeast. Deletion of sequences at either the amino or carboxyl terminus of BRF1 gave both temperature- and cold-sensitive phenotypes. These temperature- and cold-sensitive strains were used to prepare extracts deficient in BRF1 activity and were tested for transcriptional activity by RNA polymerases I, II, and III in vitro. BRF1-deficient extracts are defective in Pol III transcription and can be reconstituted for Pol III transcription by the addition of recombinant BRF1. Western analysis shows that BRF1 is present in TFIIIB but not the TFIIIC fraction, suggesting that it is a component of TFIIIB. We propose that BRF1 plays a role in Pol III initiation analogous to the role played by TFIIB for Pol II in its interaction with TBP and polymerase. The identification of a Pol III-specific TFIIB-like factor extends the previously noted similarity of transcriptional initiation by the three nuclear polymerases.
一个抑制基因被鉴定出来，该基因在高拷贝数时能挽救酵母TATA结合蛋白（TBP）中的温度敏感突变。抑制作用具有等位基因特异性，因为该抑制基因不能挽救另一个TBP突变体的温度敏感表型。这个抑制基因编码一个596个氨基酸的蛋白质，其氨基末端的一半与Pol II特异性因子TFIIB同源。这个被称为BRF1的基因的破坏表明它对酵母的生长是必需的。BRF1氨基或羧基末端序列的缺失产生了温度敏感和冷敏感的表型。这些温度敏感和冷敏感菌株被用于制备缺乏BRF1活性的提取物，并通过RNA聚合酶I、II和III在体外测试其转录活性。缺乏BRF1的提取物在Pol III转录方面存在缺陷，通过添加重组BRF1可以恢复其Pol III转录活性。蛋白质免疫印迹分析表明BRF1存在于TFIIIB中，但不存在于TFIIIC组分中，这表明它是TFIIIB的一个组分。我们提出BRF1在Pol III起始过程中发挥的作用类似于TFIIB在Pol II与TBP和聚合酶相互作用中所起的作用。一个Pol III特异性的TFIIB样因子的鉴定扩展了之前所指出的三种核聚合酶转录起始的相似性。
