Influenza polymerase (FluPol) carries out both viral transcription and replication using the same viral genome segment as a template to yield distinct end products. However, it remains largely unclear how FluPol synthesizes transcripts containing poly (A) tails during transcription termination, while producing fully complementary products during replication termination. In this study, through structural analysis combined with cell-based and biochemical assays, we identified that the PB1 Leu675/Asn676 and PB2 Arg38 residues of FluPol are critical for transcription termination and polyadenylation. During transcription termination, these three residues adopt the PB1 Leu675/Asn676down and PB2 Arg38out conformations, with their side chains positioned against the G12 and G14 residues of the RNA template at the 5' end. These steric hindrances block template translocation and facilitate FluPol 'stuttering' at U17, which is required for viral messenger RNA polyadenylation. Importantly, both structural analysis and mutational studies suggest that this specific conformation of these residues is unique to the transcription termination state. Overall, our findings provide novel insights into the mechanisms by which FluPol generates distinct 3' end products during transcription and replication termination.

中文翻译：

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诱变研究表明，多聚腺苷酸化过程中流感聚合酶停滞的机制。


流感聚合酶 （FluPol） 使用相同的病毒基因组片段作为模板进行病毒转录和复制，以产生不同的最终产物。然而，目前尚不清楚 FluPol 如何在转录终止期间合成包含多聚 （A） 尾的转录本，同时在复制终止期间产生完全互补的产物。在这项研究中，通过结构分析结合基于细胞和生化的测定，我们确定了 FluPol 的 PB1 Leu675/Asn676 和 PB2 Arg38 残基对转录终止和聚腺苷酸化至关重要。在转录终止过程中，这三个残基采用 PB1 Leu675/Asn676down 和 PB2 Arg38out 构象，它们的侧链位于 RNA 模板的 5' 末端的 G12 和 G14 残基上。这些空间位阻了模板易位并促进了 FluPol 在 U17 处“卡顿”，这是病毒信使 RNA 多聚腺苷酸化所必需的。重要的是，结构分析和突变研究表明，这些残基的这种特异性构象是转录终止状态所独有的。总体而言，我们的研究结果为 FluPol 在转录和复制终止过程中产生不同 3' 终产物的机制提供了新的见解。