RNA recognition by the DNA end-binding Ku heterodimer
RNA recognition by the DNA end-binding Ku heterodimer
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DOI：
10.1261/rna.038703.113
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发表时间：
2013-06-01
期刊：
影响因子：
4.5
通讯作者：
Cech, Thomas R.
中科院分区：
文献类型：
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作者：
Dalby, Andrew B.;Goodrich, Karen J.;Cech, Thomas R.
大多数核酸结合蛋白选择性地结合DNA或RNA，但不能同时结合两种核酸。酿酒酵母Ku异源二聚体的不同寻常之处在于它有两种非常不同的生物学相关结合模式：(1)Ku是一种序列非特异性的双链DNA末端结合蛋白，在非同源末端连接和端粒封顶中发挥重要作用；(2)Ku与出芽酵母端粒酶的RNA亚基TLC1的一个特定的茎环结合，是这种核蛋白酶正确核定位所必需的。TLC1RNA结合和dsDNA结合是互斥的，因此它们可能是由Ku上的同一个位点介导的。虽然Ku与dsDNA的结合已经得到了很好的研究，但关于RNA发夹的哪些特征能够使Ku特异性识别的研究要少得多。为了解决这个问题，我们利用硫代足迹技术用单核苷酸分辨率定位了TLC1发夹的Ku结合位点，利用化学修饰确定了发夹二级结构中的一个未知基序，并对茎环进行了突变，以确定RNA中允许Ku结合的关键元件。最后，我们提供了在附加的芽期酵母端粒酶RNA中存在Ku结合位点的证据，并讨论了RNA结合是Ku异源二聚体的保守功能的可能性。
Most nucleic acid-binding proteins selectively bind either DNA or RNA, but not both nucleic acids. The Saccharomyces cerevisiae Ku heterodimer is unusual in that it has two very different biologically relevant binding modes: (1) Ku is a sequence-nonspecific double-stranded DNA end-binding protein with prominent roles in nonhomologous end-joining and telomeric capping, and (2) Ku associates with a specific stem-loop of TLC1, the RNA subunit of budding yeast telomerase, and is necessary for proper nuclear localization of this ribonucleoprotein enzyme. TLC1 RNA-binding and dsDNA-binding are mutually exclusive, so they may be mediated by the same site on Ku. Although dsDNA binding by Ku is well studied, much less is known about what features of an RNA hairpin enable specific recognition by Ku. To address this question, we localized the Ku-binding site of the TLC1 hairpin with single-nucleotide resolution using phosphorothioate footprinting, used chemical modification to identify an unpredicted motif within the hairpin secondary structure, and carried out mutagenesis of the stem-loop to ascertain the critical elements within the RNA that permit Ku binding. Finally, we provide evidence that the Ku-binding site is present in additional budding yeast telomerase RNAs and discuss the possibility that RNA binding is a conserved function of the Ku heterodimer.