Szentirmay M N, Sawadogo M
Department of Molecular Genetics, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030.
EMBO J. 1993 Dec;12(12):4677-84. doi: 10.1002/j.1460-2075.1993.tb06156.x.
Previous studies have revealed that the in vitro synthesis of reinitiated transcripts by RNA polymerase II requires an additional activity, designated reinitiation transcription factor (RTF), which is distinct from all of the general class II initiation factors. While further characterizing this activity, it was found that RTF displays properties indistinguishable from those of the RNA polymerase II elongation factor SII. In addition, Western blot analysis using SII-specific antibodies revealed that human SII is a major component in purified RTF preparations. The functional equivalence of the two proteins was established using recombinant SII, which proved fully capable of substituting for RTF in the reinitiation assay. In these reconstituted reactions, transcription complexes resulting from reinitiation events required SII to proceed through a 400 bp G-free cassette, while complexes resulting from the first round of initiations were SII-independent. Reinitiations can take place in the absence of SII; however, addition of the elongation factor is essential for full extension of the reinitiated transcripts. These results suggest that events taking place at the promoter (e.g. first-round initiations versus reinitiations) can create marked differences in the properties of RNA polymerase II elongation complexes.
先前的研究表明，RNA聚合酶II体外合成重新起始的转录本需要一种额外的活性，称为重新起始转录因子（RTF），它不同于所有II类通用起始因子。在进一步表征这种活性时，发现RTF表现出与RNA聚合酶II延伸因子SII无法区分的特性。此外，使用SII特异性抗体进行的蛋白质印迹分析表明，人SII是纯化的RTF制剂中的主要成分。使用重组SII确定了这两种蛋白质的功能等效性，重组SII在重新起始测定中被证明完全能够替代RTF。在这些重构反应中，重新起始事件产生的转录复合物需要SII才能通过400 bp的无G盒，而第一轮起始产生的复合物则不依赖SII。重新起始可以在没有SII的情况下发生；然而，添加延伸因子对于重新起始转录本的完全延伸至关重要。这些结果表明，在启动子处发生的事件（例如第一轮起始与重新起始）可以在RNA聚合酶II延伸复合物的特性上产生显著差异。
