Kang J J, Yokoi T J, Holland M J
Department of Biological Chemistry, School of Medicine, University of California, Davis 95616-8635, USA.
J Biol Chem. 1995 Dec 1;270(48):28723-32. doi: 10.1074/jbc.270.48.28723.
The 190-base pair (bp) rDNA enhancer within the intergenic spacer sequences of Saccharomyces cerevisiae rRNA cistrons activates synthesis of the 35S-rRNA precursor about 20-fold in vivo (Mestel,, R., Yip, M., Holland, J. P., Wang, E., Kang, J., and Holland, M. J. (1989) Mol. Cell. Biol. 9, 1243-1254). We now report identification and analysis of transcriptional activities mediated by three cis-acting sites within a 90-bp portion of the rDNA enhancer designated the modulator region. In vivo, these sequences mediated termination of transcription by RNA polymerase I and potentiated the activity of the rDNA enhancer element. Two trans-acting factors, REB1 and REB2, bind independently to sites within the modulator region (Morrow, B. E., Johnson, S. P., and Warner, J. R. (1989) J. Biol. Chem. 264, 9061-9068). We show that REB2 is identical to the ABF1 protien. Site-directed mutagenesis of REB1 and ABF1 binding sites demonstrated uncoupling of RNA polymerase I-dependent termination from transcriptional activation in vivo. We conclude that REB1 and ABF1 are required for RNA polymerase I-dependent termination and enhancer function, respectively, Since REB1 and ABF1 proteins also regulate expression of class II genes and other nuclear functions, our results suggest further similarities between RNA polymerase I and II regulatory mechanisms. Two rDNA enhancers flanking a rDNA minigene stimulated RNA polymerase I transcription in a "multiplicative" fashion. Deletion mapping analysis showed that similar cis-acting sequences were required for enhancer function when positioned upstream or downstream from a rDNA minigene.
酿酒酵母rRNA顺反子基因间隔序列中的190个碱基对（bp）的rDNA增强子在体内可使35S - rRNA前体的合成激活约20倍（梅斯特尔，R.，叶，M.，霍兰德，J.P.，王，E.，康，J.，以及霍兰德，M.J.（1989年）《分子与细胞生物学》9，1243 - 1254）。我们现在报告对rDNA增强子中一个90 bp部分（称为调节区）内三个顺式作用位点介导的转录活性的鉴定和分析。在体内，这些序列介导RNA聚合酶I的转录终止，并增强rDNA增强子元件的活性。两种反式作用因子，REB1和REB2，独立结合到调节区内的位点（莫罗，B.E.，约翰逊，S.P.，以及华纳，J.R.（1989年）《生物化学杂志》264，9061 - 9068）。我们表明REB2与ABF1蛋白相同。对REB1和ABF1结合位点的定点诱变表明，在体内RNA聚合酶I依赖性终止与转录激活解偶联。我们得出结论，REB1和ABF1分别是RNA聚合酶I依赖性终止和增强子功能所必需的。由于REB1和ABF1蛋白也调节II类基因的表达和其他核功能，我们的结果表明RNA聚合酶I和II调节机制之间存在进一步的相似性。一个rDNA小基因两侧的两个rDNA增强子以“倍增”方式刺激RNA聚合酶I转录。缺失定位分析表明，当位于rDNA小基因的上游或下游时，增强子功能需要相似的顺式作用序列。
