Pritlove D C, Poon L L, Fodor E, Sharps J, Brownlee G G
Sir William Dunn School of Pathology, University of Oxford, United Kingdom.
J Virol. 1998 Feb;72(2):1280-6. doi: 10.1128/JVI.72.2.1280-1286.1998.
Here we report the development of two independent assays which demonstrate for the first time that exogenous model RNA templates based on influenza virus virion RNA (vRNA) are transcribed in vitro to produce polyadenylated mRNA. We investigated the activities of mutated templates with known polymerase binding properties to test our model that polyadenylation occurs when a polymerase complex, which is bound to conserved 5' sequences of vRNA, prevents read-through of the U track at which polyadenylation subsequently occurs by reiterative copying. Mutated templates with perturbed polymerase binding sites (i.e., a deletion mutant lacking the first 4 5' residues and a U-->A point mutant at the third residue) initiated transcription in the in vitro assay but failed to produce polyadenylated transcripts, whereas an A-->U point mutant at the fourth residue, which retained polymerase binding properties similar to those of the wild type, produced polyadenylated transcripts. Our results show that nucleotides within the conserved 5' sequence are required for polyadenylation and support the hypothesis that polymerase binding to 5' sequences of the template is required for mRNA synthesis.
在此，我们报告了两种独立检测方法的开发情况，这两种方法首次证明，基于流感病毒病毒粒子RNA（vRNA）的外源模型RNA模板在体外被转录以产生多聚腺苷酸化的mRNA。我们研究了具有已知聚合酶结合特性的突变模板的活性，以检验我们的模型，即当与vRNA保守5'序列结合的聚合酶复合物通过反复复制阻止随后发生多聚腺苷酸化的U序列通读时，就会发生多聚腺苷酸化。具有受干扰聚合酶结合位点的突变模板（即缺少前4个5'残基的缺失突变体和第3个残基处的U→A点突变体）在体外检测中启动了转录，但未能产生多聚腺苷酸化转录本，而第4个残基处的A→U点突变体保留了与野生型相似的聚合酶结合特性，产生了多聚腺苷酸化转录本。我们的结果表明，保守5'序列内的核苷酸是多聚腺苷酸化所必需的，并支持这样的假设，即模板的mRNA合成需要聚合酶与5'序列结合。
