CEITEC-Central European Institute of Technology, Masaryk University, Kamenice 5, CZ-62500 Brno, Czech Republic.
Nucleic Acids Res. 2012 Jul;40(12):5679-93. doi: 10.1093/nar/gks223. Epub 2012 Mar 8.
Trf4/5p-Air1/2p-Mtr4p polyadenylation complex (TRAMP) is an essential component of nuclear RNA surveillance in yeast. It recognizes a variety of nuclear transcripts produced by all three RNA polymerases, adds short poly(A) tails to aberrant or unstable RNAs and activates the exosome for their degradation. Despite the advances in understanding the structural features of the isolated complex subunits or their fragments, the details of complex assembly, RNA recognition and exosome activation remain poorly understood. Here we provide the first understanding of the RNA binding mode of the complex. We show that Air2p is an RNA-binding subunit of TRAMP. We identify the zinc knuckles (ZnK) 2, 3 and 4 as the RNA-binding domains, and reveal the essentiality of ZnK4 for TRAMP4 polyadenylation activity. Furthermore, we identify Air2p as the key component of TRAMP4 assembly providing bridging between Mtr4p and Trf4p. The former is bound via the N-terminus of Air2p, while the latter is bound via ZnK5, the linker between ZnK4 and 5 and the C-terminus of the protein. Finally, we uncover the RNA binding part of the Mtr4p arch, the KOW domain, as the essential component for TRAMP-mediated exosome activation.
TRAMP 多聚腺苷酸化复合物（TRAMP）是酵母核 RNA 监测的必需组成部分。它识别由所有三种 RNA 聚合酶产生的多种核转录本，向异常或不稳定的 RNA 添加短 poly(A)尾巴，并激活外切体进行降解。尽管在理解分离的复合物亚基或其片段的结构特征方面取得了进展，但复合物组装、RNA 识别和外切体激活的细节仍知之甚少。在这里，我们首次了解了该复合物的 RNA 结合模式。我们表明 Air2p 是 TRAMP 的 RNA 结合亚基。我们确定锌指 (ZnK) 2、3 和 4 是 RNA 结合结构域，并揭示了 ZnK4 对 TRAMP4 多聚腺苷酸化活性的必需性。此外，我们确定 Air2p 是 TRAMP4 组装的关键组成部分，为 Mtr4p 和 Trf4p 提供桥接。前者通过 Air2p 的 N 端结合，而后者通过 ZnK5、ZnK4 和 5 之间的连接子以及蛋白质的 C 端结合。最后，我们揭示了 Mtr4p 拱的 RNA 结合部分，即 KOW 结构域，作为 TRAMP 介导的外切体激活的必需组成部分。
