Variation in ATP requirement during influenza virus transcription
Variation in ATP requirement during influenza virus transcription
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DOI：
10.1099/0022-1317-79-5-1033
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发表时间：
1998-05-01
影响因子：
3.8
通讯作者：
Ruigrok, RWH
中科院分区：
文献类型：
--
作者：
Klumpp, K;Ford, MJ;Ruigrok, RWH
在体外转录反应期间，研究了甲型流感病毒RNA依赖性RNA聚合酶的ATP需求。在完全转录反应中，ATP的Km值比其他NTPs的Km值高10倍。然而，在转录延伸过程中，ATP的Km值与其他NTPs的Km值一样低，这表明在转录起始过程中对ATP的特殊要求。转录起始反应的RNA产物的凝胶分析表明，AMP到新生RNA中的掺入在相对于模板RNA的位置4、6和7处比在位置5处更有效。聚合酶产生短的，失败的成绩单与长度对应的位置3和4相对于模板，但从来没有位置5或更长。这些结果表明，在位置5处掺入AMP诱导甲型流感病毒聚合酶经历从转录起始到延伸复合物的转变。聚合酶复合物的这种功能变化而不是ATP β-γ键水解的需要是转录早期ATP的特别高的K-m的最可能原因。这一结论得到以下事实的支持：ATP类似物ATP γ S [腺苷5 '-O-（3-硫代三磷酸）]可以在体外转录反应中有效地取代ATP，并且在转录起始和延伸之间显示出相当的Km下降。
The ATP requirement of influenza A virus RNA-dependent RNA polymerase was studied during in vitro transcription reactions. In complete transcription reactions, the K-m for ATP was 10-fold higher than the K-m values for the other NTPs. However, during transcription elongation the K-m for ATP was as low as the K-m values for the other NTPs, suggesting a special requirement for ATP during transcription initiation. Gel analysis of RNA products of transcription initiation reactions showed that the incorporation of AMP into nascent RNA was more efficient at positions 4, 6 and 7 relative to the template RNA than at position 5. The polymerase produced short, abortive transcripts with lengths corresponding to positions 3 and 4 relative to the template but never to position 5 or longer. These results suggest that incorporation of AMP at position 5 induces the influenza A virus polymerase to go through a transition from a transcription initiation to an elongation complex. This functional change of the polymerase complex rather than a requirement for ATP beta-gamma bond hydrolysis is the most likely reason for the particularly high K-m for ATP during the early phase of transcription. This conclusion is supported by the fact that the ATP analogue ATP gamma S [adenosine 5'-O-(3-thiotriphosphate)] can efficiently replace ATP in in vitro transcription reactions and shows a comparable drop of K-m between transcription initiation and elongation.