Northeastern University, Department of Physics, Boston, MA02115, USA.
The Laboratory of Molecular and Cellular Biology, NIDDK, NIH, Bethesda, MD 20892, USA.
Nucleic Acids Res. 2022 Aug 26;50(15):8690-8699. doi: 10.1093/nar/gkac628.
Long interspersed nuclear element 1 (L1) parasitized most vertebrates and constitutes ∼20% of the human genome. It encodes ORF1p and ORF2p which form an L1-ribonucleoprotein (RNP) with their encoding transcript that is copied into genomic DNA (retrotransposition). ORF1p binds single-stranded nucleic acid (ssNA) and exhibits NA chaperone activity. All vertebrate ORF1ps contain a coiled coil (CC) domain and we previously showed that a CC-retrotransposition null mutant prevented formation of stably bound ORF1p complexes on ssNA. Here, we compared CC variants using our recently improved method that measures ORF1p binding to ssDNA at different forces. Bound proteins decrease ssDNA contour length and at low force, retrotransposition-competent ORF1ps (111p and m14p) exhibit two shortening phases: the first is rapid, coincident with ORF1p binding; the second is slower, consistent with formation of tightly compacted complexes by NA-bound ORF1p. In contrast, two retrotransposition-null CC variants (151p and m15p) did not attain the second tightly compacted state. The C-terminal half of the ORF1p trimer (not the CC) contains the residues that mediate NA-binding. Our demonstrating that the CC governs the ability of NA-bound retrotransposition-competent trimers to form tightly compacted complexes reveals the biochemical phenotype of these coiled coil mutants.
长散布核元件 1（L1）寄生在大多数脊椎动物中，构成人类基因组的约 20%。它编码 ORF1p 和 ORF2p，它们与编码的转录本形成 L1-核糖核蛋白（RNP），该转录本被复制到基因组 DNA 中（逆转录转座）。ORF1p 结合单链核酸（ssNA）并表现出 NA 分子伴侣活性。所有脊椎动物的 ORF1ps 都包含一个卷曲螺旋（CC）结构域，我们之前的研究表明，CC-逆转录转座缺失突变体阻止了稳定结合的 ORF1p 复合物在 ssNA 上的形成。在这里，我们使用我们最近改进的方法比较了 CC 变体，该方法测量了 ORF1p 在不同力下与 ssDNA 的结合。结合蛋白会缩短 ssDNA 的轮廓长度，在低力下，具有逆转录转座能力的 ORF1ps（111p 和 m14p）表现出两个缩短阶段：第一个阶段是快速的，与 ORF1p 结合同时发生；第二个阶段较慢，与 NA 结合的 ORF1p 形成紧密压缩的复合物一致。相比之下，两个逆转录转座缺失的 CC 变体（151p 和 m15p）没有达到第二个紧密压缩状态。ORF1p 三聚体的 C 末端（不是 CC）包含介导 NA 结合的残基。我们证明 CC 控制了 NA 结合的逆转录转座能力的三聚体形成紧密压缩复合物的能力，揭示了这些卷曲螺旋突变体的生化表型。
