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首页> 外文期刊>Biochemistry >ALANINE-SCANNING MUTAGENESIS OF HUMAN TRANSCRIPT ELONGATION FACTOR TFIIS
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ALANINE-SCANNING MUTAGENESIS OF HUMAN TRANSCRIPT ELONGATION FACTOR TFIIS

机译：人体转录延伸因子TFIIS的丙氨酸扫描诱变

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摘要

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 IT 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.
机译：TFIIS是一种转录延伸因子，可与RNA聚合酶II结合，并通过诱导新生转录本3'端附近的切割，使其通过多种转录阻断进行转录。尽管这种裂解反应在TFIIS重新激活转录过程中起关键作用，但TFIIS通过RNA聚合酶IT促进通读的确切机制尚未完全了解。因此，我们对TFIIS（Delta TFIIS）的C端一半进行了系统诱变，以评估该区域中带电残基的贡献，以诱导转录物切割并促进体外通读。通过在HeLa TFIIS C末端一半的带电残基簇中用每个氨基酸替换丙氨酸来构建22个Delta TFIIS丙氨酸扫描突变体。在体外使用阻滞于延伸的阻滞的RNA聚合酶II三元延伸复合物测试了诱导这些突变体的转录物切割和通读的能力。丙氨酸扫描诱变分析可以鉴定对TFIIS活性重要的区域或残基。许多突变体的裂解和通读活性均降低。但是，在某些情况下，这些活动减少之间没有简单的关联。

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