Laboratory of Structural Biochemistry, Freie Universität Berlin, Takustraβe 6, D-14195 Berlin, Germany.
Medizinische Physik und Biophysik, Charité - Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany.
Nat Microbiol. 2017 Apr 28;2:17062. doi: 10.1038/nmicrobiol.2017.62.
λN-mediated processive antitermination constitutes a paradigmatic transcription regulatory event, during which phage protein λN, host factors NusA, NusB, NusE and NusG, and an RNA nut site render elongating RNA polymerase termination-resistant. The structural basis of the process has so far remained elusive. Here we describe a crystal structure of a λN-NusA-NusB-NusE-nut site complex and an electron cryo-microscopic structure of a complete transcription antitermination complex, comprising RNA polymerase, DNA, nut site RNA, all Nus factors and λN, validated by crosslinking/mass spectrometry. Due to intrinsic disorder, λN can act as a multiprotein/RNA interaction hub, which, together with nut site RNA, arranges NusA, NusB and NusE into a triangular complex. This complex docks via the NusA N-terminal domain and the λN C-terminus next to the RNA exit channel on RNA polymerase. Based on the structures, comparative crosslinking analyses and structure-guided mutagenesis, we hypothesize that λN mounts a multipronged strategy to reprogram the transcriptional machinery, which may include (1) the λN C terminus clamping the RNA exit channel, thus stabilizing the DNA:RNA hybrid; (2) repositioning of NusA and RNAP elements, thus redirecting nascent RNA and sequestering the upstream branch of a terminator hairpin; and (3) hindering RNA engagement of termination factor ρ and/or obstructing ρ translocation on the transcript.
λN 介导的连续抗终止是一个典型的转录调控事件，在此过程中，噬菌体蛋白 λN、宿主因子 NusA、NusB、NusE 和 NusG 以及 RNA 营养位点使正在延伸的 RNA 聚合酶具有抗终止的能力。到目前为止，该过程的结构基础仍不清楚。在这里，我们描述了一个 λN-NusA-NusB-NusE-营养位点复合物的晶体结构和一个完整转录抗终止复合物的电子冷冻显微镜结构，该复合物由 RNA 聚合酶、DNA、营养位点 RNA、所有 Nus 因子和 λN 组成，并通过交联/质谱进行了验证。由于固有无序，λN 可以作为一个多蛋白/RNA 相互作用中心，与营养位点 RNA 一起，将 NusA、NusB 和 NusE 排列成一个三角形复合物。该复合物通过 NusA N 端结构域和 λN C 端与 RNA 聚合酶的 RNA 出口通道相邻的位置对接。基于结构、比较交联分析和结构指导的突变，我们假设 λN 采用了一种多管齐下的策略来重新编程转录机制，这可能包括：(1) λN C 端夹住 RNA 出口通道，从而稳定 DNA:RNA 杂交；(2) 重新定位 NusA 和 RNA 聚合酶元件，从而重新引导新生 RNA 并隔离终止子发夹的上游分支；(3) 阻碍终止因子 ρ 与 RNA 的结合，和/或阻止 ρ 在转录本上的易位。
