Martin Sandra L, Branciforte Dan, Keller David, Bain David L
Department of Cell and Developmental Biology and Program in Molecular Biology, University of Colorado School of Medicine, 4200 East Ninth Avenue, Denver, CO 80262, USA.
Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):13815-20. doi: 10.1073/pnas.2336221100. Epub 2003 Nov 13.
Two proteins are encoded by the mammalian retrotransposon long interspersed nuclear element 1 (LINE-1 or L1); both are essential for retrotransposition. The function of the protein encoded by the 5'-most ORF, ORF1p, is incompletely understood, although the ORF1p from mouse L1 is known to bind single-stranded nucleic acids and function as a nucleic acid chaperone. ORF1p self-associates by means of a long coiled-coil domain in the N-terminal region of the protein, and the basic, C-terminal region (C-1/3 domain) contains the nucleic acid binding activity. The full-length and C-1/3 domains of ORF1p were purified to near homogeneity then analyzed by gel filtration chromatography and analytical ultracentrifugation. Both proteins were structurally homogeneous and asymmetric in solution, with the full-length version forming a stable trimer and the C-1/3 domain remaining a monomer. Examination of the full-length protein by atomic force microscopy revealed an asymmetric dumbbell shape, congruent with the chromatography and ultracentrifugation results. These structural features are compatible with the nucleic acid binding and chaperone activities of L1 ORF1p and offer further insight into the functions of this unique protein during LINE-1 retrotransposition.
哺乳动物逆转录转座子长散在核元件1（LINE-1或L1）编码两种蛋白质；这两种蛋白质对于逆转录转座都是必不可少的。虽然已知来自小鼠L1的ORF1p能结合单链核酸并作为核酸伴侣发挥作用，但对5'-最末端开放阅读框（ORF）编码的蛋白质ORF1p的功能仍未完全了解。ORF1p通过蛋白质N端区域的一个长卷曲螺旋结构域进行自我缔合，而碱性的C端区域（C-1/3结构域）具有核酸结合活性。将ORF1p的全长和C-1/3结构域纯化至接近均一状态，然后通过凝胶过滤色谱法和分析超速离心法进行分析。两种蛋白质在溶液中结构均一且不对称，全长形式形成稳定的三聚体，而C-1/3结构域则保持单体状态。通过原子力显微镜对全长蛋白质的检查显示出不对称哑铃形状，这与色谱法和超速离心结果一致。这些结构特征与L1 ORF1p的核酸结合和伴侣活性相符，并为深入了解这种独特蛋白质在LINE-1逆转录转座过程中的功能提供了进一步的线索。
