Kaiser M W, Brow D A
Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison 53706-1532, USA.
J Biol Chem. 1995 May 12;270(19):11398-405. doi: 10.1074/jbc.270.19.11398.
The B block promoter element is the primary binding site for the RNA polymerase III transcription initiation factor TFIIIC. It is always located within the transcript coding region, except in the Saccharomyces cerevisiae U6 RNA gene (SNR6), in which the B block lies 120 base pairs downstream of the terminator. We have exploited the unique location of the SNR6 B block to examine the sequence specificity of its interaction with TFIIIC. The in vitro and in vivo effects of all possible single base pair substitutions in the 9-base pair core of the B block were determined. Five mutant alleles are recessive lethal when present at a low copy number; these alleles identify crucial contacts between TFIIIC and the B block promoter element. Transcript analysis reveals that lethal B block substitutions reduce U6 RNA synthesis at least 10-fold in vivo and 20-fold in vitro. One viable B block mutant strain has one-third the wild type amount of U6 RNA and exhibits reduced levels of the U4-U6 RNA complex required for spliceosome assembly. The locations of lethal single and double point mutations leads us to propose that two domains of TFIIIC contact overlapping sites on the B block element.
B 框启动子元件是RNA聚合酶III转录起始因子TFIIIC的主要结合位点。它总是位于转录本编码区内，酿酒酵母U6 RNA基因（SNR6）除外，在该基因中，B框位于终止子下游120个碱基对处。我们利用SNR6 B框的独特位置来研究其与TFIIIC相互作用的序列特异性。确定了B框9个碱基对核心区内所有可能的单碱基对替换的体外和体内效应。当以低拷贝数存在时，五个突变等位基因是隐性致死的；这些等位基因确定了TFIIIC与B框启动子元件之间的关键接触点。转录分析表明，致死性B框替换在体内使U6 RNA合成减少至少10倍，在体外减少20倍。一个可存活的B框突变菌株的U6 RNA量为野生型的三分之一，并且剪接体组装所需的U4-U6 RNA复合物水平降低。致死性单碱基和双碱基点突变的位置使我们提出，TFIIIC的两个结构域与B框元件上的重叠位点接触。
