An in vitro fluorescence based study of initiation of RNA synthesis by influenza B polymerase.
An in vitro fluorescence based study of initiation of RNA synthesis by influenza B polymerase.
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DOI：
10.1093/nar/gkx043
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发表时间：
2017-04-07
影响因子：
14.9
通讯作者：
Cusack S
中科院分区：
文献类型：
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作者：
Reich S;Guilligay D;Cusack S
流感病毒聚合酶通过互补RNA中间体（cRNA）复制，并转录八个病毒RNA（vRNA）基因组片段。为了启动RNA合成，它与vRNA或cRNA的保守的5 ′端和3 ′端（“启动子”）结合。在远端启动子区域中的5 ′-3 ′碱基配对对于将模板RNA定位在聚合酶活性位点是必需的，如具有穿过模板入口隧道的3 ′末端的新晶体结构所示。我们开发了荧光偏振测定法，以定量与vRNA或cRNA启动子结合的重组乙型流感B聚合酶引发的帽引发（转录）或未引发（复制）RNA合成的起始。限速步骤是形成引发的起始复合物，其中最低限度需要ApG来稳定活性位点内模板的3 ′端。与vRNA启动子结合的聚合酶在末端启动RNA合成，而cRNA启动子以显著较低的速率指导内部起始。延伸的进展需要破坏启动子5 ′-3 ′碱基配对区域，并通过出现的模板产物碱基对进行有利的补偿。RNA合成测定适用于聚合酶抑制剂的高通量筛选。在一项初步研究中，我们发现，在cRNA启动子的启动是不寻常的容易受到抑制的2 NF-2 β dNTPs。
Influenza polymerase replicates, via a complementary RNA intermediate (cRNA), and transcribes the eight viral RNA (vRNA) genome segments. To initiate RNA synthesis it is bound to the conserved 5΄ and 3΄ extremities of the vRNA or cRNA (the ‘promoter’). 5΄-3΄ base-pairing in the distal promoter region is essential to position the template RNA at the polymerase active site, as shown by a new crystal structure with the 3΄ end threading through the template entry tunnel. We develop fluorescence polarization assays to quantify initiation of cap-primed (transcription) or unprimed (replication) RNA synthesis by recombinant influenza B polymerase bound to the vRNA or cRNA promoter. The rate-limiting step is formation of a primed initiation complex with minimally ApG required to stabilize the 3΄ end of the template within the active-site. Polymerase bound to the vRNA promoter initiates RNA synthesis terminally, while the cRNA promoter directs internal initiation at a significantly lower rate. Progression to elongation requires breaking the promoter 5΄-3΄ base-pairing region and favourable compensation by the emerging template-product base-pairs. The RNA synthesis assay is adaptable to high-throughput screening for polymerase inhibitors. In a pilot study, we find that initiation at the cRNA promoter is unusually susceptible to inhibition by 2΄F-2΄dNTPs.