Archambault J, Jansma D B, Kawasoe J H, Arndt K T, Greenblatt J, Friesen J D
Banting and Best Department of Medical Research and Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5G 1X8, Canada.
J Bacteriol. 1998 May;180(10):2590-8. doi: 10.1128/JB.180.10.2590-2598.1998.
Mutations that increase the low-level transcription of the Saccharomyces cerevisiae HIS4 gene, which results from deletion of the genes encoding transcription factors BAS1, BAS2, and GCN4, were isolated previously in SIT1 (also known as RPO21, RPB1, and SUA8), the gene encoding the largest subunit of RNA polymerase II (RNAPII). Here we show that sit1 substitutions cluster in two conserved regions of the enzyme which form part of the active site. Six sit1 mutations, affect region F, a region that is involved in transcriptional elongation and in resistance to alpha-aminatin. Four sit1 substitutions lie in another region involved in transcriptional elongation, region D, which binds Mg2+ ions essential for RNA catalysis. One region D substitution is lethal unless suppressed by a substitution in region G and interacts genetically with PPR2, the gene encoding transcription elongation factor IIS. Some sit1 substitutions affect the selection of transcriptional start sites at the CYC1 promoter in a manner reminiscent of that of sua8 (sua stands for suppression of upstream ATG) mutations. Together with previous findings which indicate that regions D and G are in close proximity to the 3' end of the nascent transcript and that region F is involved in the translocation process, our results suggest that transcriptional activation by the sit1 mutations results from alteration of the RNAPII active center.
先前在SIT1（也称为RPO21、RPB1和SUA8）中分离出了一些突变，这些突变会增加酿酒酵母HIS4基因的低水平转录，该基因的低水平转录是由于编码转录因子BAS1、BAS2和GCN4的基因缺失所致，SIT1基因编码RNA聚合酶II（RNAPII）的最大亚基。在这里我们表明，sit1替代突变集中在该酶的两个保守区域，这两个区域构成了活性位点的一部分。六个sit1突变影响区域F，该区域参与转录延伸和对α-鹅膏菌素的抗性。四个sit1替代突变位于另一个参与转录延伸的区域，即区域D，该区域结合RNA催化所必需的Mg2+离子。区域D的一个替代突变是致死性的，除非被区域G中的一个替代突变所抑制，并且它与编码转录延伸因子IIS的PPR2基因发生遗传相互作用。一些sit1替代突变以类似于sua8（sua代表上游ATG的抑制）突变的方式影响CYC1启动子处转录起始位点的选择。结合先前的研究结果，即区域D和G靠近新生转录本的3'末端，以及区域F参与易位过程，我们的结果表明，sit1突变引起的转录激活是由RNAPII活性中心的改变导致的。
