Zhao Yu, McIntosh Kerri B, Rudra Dipayan, Schawalder Stephan, Shore David, Warner Jonathan R
Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, USA.
Mol Cell Biol. 2006 Jul;26(13):4853-62. doi: 10.1128/MCB.02367-05.
The ribosomal protein genes of Saccharomyces cerevisiae, responsible for nearly 40% of the polymerase II transcription initiation events, are characterized by the constitutive tight binding of the transcription factor Rap1. Rap1 binds at many places in the yeast genome, including glycolytic enzyme genes, the silent MAT loci, and telomeres, its specificity arising from specific cofactors recruited at the appropriate genes. At the ribosomal protein genes two such cofactors have recently been identified as Fhl1 and Ifh1. We have now characterized the interaction of these factors at a bidirectional ribosomal protein promoter by replacing the Rap1 sites with LexA operator sites. LexA-Gal4(AD) drives active transcription at this modified promoter, although not always at the correct initiation site. Tethering Rap1 to the promoter neither drives transcription nor recruits Fhl1 or Ifh1, showing that Rap1 function requires direct DNA binding. Tethering Fhl1 also fails to activate transcription, even though it does recruit Ifh1, suggesting that Fhl1 does more than simply provide a platform for Ifh1. Tethering Ifh1 to the promoter leads to low-level transcription, at the correct initiation sites. Remarkably, activation by tethered LexA-Gal4(AD) is strongly reduced when TOR kinase is inhibited by rapamycin. Thus, TOR can act independently of Fhl1/Ifh1 at ribosomal protein promoters. We also show that, in our strain background, the response of ribosomal protein promoters to TOR inhibition is independent of the Ifh1-related protein Crf1, indicating that the role of this corepressor is strain specific. Fine-structure chromatin mapping of several ribosomal protein promoters revealed that histones are essentially absent from the Rap1 sites, while Fhl1 and Ifh1 are coincident with each other but distinct from Rap1.
酿酒酵母的核糖体蛋白基因负责近40%的聚合酶II转录起始事件，其特征是转录因子Rap1的组成型紧密结合。Rap1在酵母基因组的许多位置结合，包括糖酵解酶基因、沉默的MAT位点和端粒，其特异性源于在适当基因处募集的特定辅因子。在核糖体蛋白基因处，最近已鉴定出两种这样的辅因子为Fhl1和Ifh1。我们现在通过用LexA操纵位点取代Rap1位点，对这些因子在双向核糖体蛋白启动子处的相互作用进行了表征。LexA-Gal4(AD)在这个修饰的启动子上驱动活性转录，尽管并不总是在正确的起始位点。将Rap1拴系到启动子上既不驱动转录，也不募集Fhl1或Ifh1，表明Rap1功能需要直接的DNA结合。拴系Fhl1也不能激活转录，即使它确实募集了Ifh1，这表明Fhl1的作用不仅仅是为Ifh1提供一个平台。将Ifh1拴系到启动子上会导致在正确的起始位点进行低水平转录。值得注意的是，当雷帕霉素抑制TOR激酶时，拴系的LexA-Gal4(AD)的激活会大大降低。因此，TOR可以在核糖体蛋白启动子上独立于Fhl1/Ifh1发挥作用。我们还表明，在我们的菌株背景下，核糖体蛋白启动子对TOR抑制的反应独立于Ifh1相关蛋白Crf1，这表明这种共抑制因子的作用是菌株特异性的。对几个核糖体蛋白启动子的精细结构染色质图谱分析表明，Rap1位点基本没有组蛋白，而Fhl1和Ifh1相互重合，但与Rap1不同。
