Roberts S, Miller S J, Lane W S, Lee S, Hahn S
Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
J Biol Chem. 1996 Jun 21;271(25):14903-9. doi: 10.1074/jbc.271.25.14903.
The yeast RNA polymerase III (pol III) general transcription factor TFIIIB is composed of three subunits; the TATA-binding protein (TBP)1, the TFIIB-related factor (BRF1), and a third factor termed TFIIIB90 or B". Here we report the purification of yeast TFIIIB90, cloning of the gene encoding TFIIIB90, and reconstitution of TFIIIB from recombinant polypeptides. The TFIIIB90 open reading frame encodes a 68-kDa polypeptide and has no obvious similarity to any other known protein sequences. The gene encoding TFIIIB90 is essential for viability of yeast. Using recombinant TFIIIB subunits, we found that TFIIIB90 interacts weakly with TBP in the absence of BRF1, and that this interaction is enhanced at least 25-fold by BRF1. In addition, TFIIIB90 showed pol III specificity as it could not interact with the pol II-specific TFIIB-TBP-DNA complex. To localize the regions of the TBP-DNA complex that interact with BRF1 and TFIIIB90, we tested whether the pol II factors TFIIA and TFIIB interfered with the binding of BRF1 and TFIIIB90 to TBP-DNA. Our results suggest that the binding sites for BRF1 and TFIIIB90 on TBP-DNA both overlap the binding sites for TFIIA and TFIIB.
酵母RNA聚合酶III（pol III）通用转录因子TFIIIB由三个亚基组成；TATA结合蛋白（TBP）1、TFIIB相关因子（BRF1）以及第三个称为TFIIIB90或B”的因子。在此，我们报告了酵母TFIIIB90的纯化、编码TFIIIB90的基因的克隆以及从重组多肽中重构TFIIIB。TFIIIB90开放阅读框编码一个68 kDa的多肽，与任何其他已知蛋白质序列均无明显相似性。编码TFIIIB90的基因对酵母的生存能力至关重要。使用重组TFIIIB亚基，我们发现，在没有BRF1的情况下，TFIIIB90与TBP的相互作用较弱，而BRF1可使这种相互作用增强至少25倍。此外，TFIIIB90表现出pol III特异性，因为它不能与pol II特异性的TFIIB - TBP - DNA复合物相互作用。为了定位TBP - DNA复合物中与BRF1和TFIIIB90相互作用的区域，我们测试了pol II因子TFIIA和TFIIB是否会干扰BRF1和TFIIIB90与TBP - DNA的结合。我们的结果表明，BRF1和TFIIIB90在TBP - DNA上的结合位点均与TFIIA和TFIIB的结合位点重叠。
