Cipres-Palacin G, Kane C M
Department of Molecular and Cell Biology, University of California, Berkeley 94720-3202, USA.
Biochemistry. 1995 Nov 21;34(46):15375-80. doi: 10.1021/bi00046a046.
TFIIS is a transcription elongation factor that binds to RNA polymerase II and allows it to transcribe through a variety of transcriptional blockages by inducing cleavage near the 3' end of the nascent transcript. Although this cleavage reaction plays a key role in the process of reactivation of transcription by TFIIS, the exact mechanism by which TFIIS promotes readthrough by RNA polymerase II is not completely understood. We therefore undertook a systematic mutagenesis of the C-terminal half of TFIIS (delta TFIIS) to evaluate the contribution of charged residues in this region to induce transcript cleavage and promote readthrough in vitro. Twenty-two delta TFIIS alanine-scanning mutants were constructed by substitution of alanine for each amino acid in clusters of charged residues in the C-terminal half of HeLa TFIIS. The ability to induce transcript cleavage and readthrough of these mutants was tested in vitro using RNA polymerase II ternary elongation complexes arrested at a block to elongation. This alanine-scanning mutagenesis analysis allowed the identification of regions or residues important for the activity of TFIIS. Many of the mutants were reduced alike in both cleavage and readthrough activities. However, in several cases there was no simple correlation between these activity reductions.
TFIIS是一种转录延伸因子，它与RNA聚合酶II结合，并通过在新生转录本3'端附近诱导切割，使RNA聚合酶II能够转录通过各种转录障碍。尽管这种切割反应在TFIIS介导的转录重新激活过程中起关键作用，但TFIIS促进RNA聚合酶II通读的具体机制尚未完全清楚。因此，我们对TFIIS的C端一半（δTFIIS）进行了系统诱变，以评估该区域带电残基在体外诱导转录本切割和促进通读方面的作用。通过将HeLa TFIIS C端一半中带电残基簇中的每个氨基酸替换为丙氨酸，构建了22个δTFIIS丙氨酸扫描突变体。使用停滞在延伸障碍处的RNA聚合酶II三元延伸复合物，在体外测试了这些突变体诱导转录本切割和通读的能力。这种丙氨酸扫描诱变分析有助于确定对TFIIS活性重要的区域或残基。许多突变体在切割和通读活性方面都同样降低。然而，在一些情况下，这些活性降低之间没有简单的相关性。
