Deletion analysis defines distinct functional domains for protein-protein and nucleic acid interactions in the ORF1 protein of mouse LINE-1
Deletion analysis defines distinct functional domains for protein-protein and nucleic acid interactions in the ORF1 protein of mouse LINE-1
复制标题
DOI：
10.1006/jmbi.2000.4182
复制
发表时间：
2000-11-17
影响因子：
5.6
通讯作者：
Weisz, JA
中科院分区：
文献类型：
--
作者：
Martin, SL;Li, JF;Weisz, JA
LINE-1或L1是哺乳动物中的非LTR反转录转座子。L1的反转录转座需要两个元件编码的蛋白质ORF1p和ORF2p的作用。ORF2p为逆转录和整合新转座的L1拷贝提供必要的酶活性，而ORF1p的确切作用尚不清楚。在人类和小鼠细胞提取物中，43 kDa ORF1p与L1 RNA共纯化为大复合物。从大肠杆菌中纯化的小鼠ORF1p在体外结合RNA和单链DNA，表现出核酸伴侣活性，并且能够进行蛋白质-蛋白质相互作用。在这项研究中，我们创建了一系列的ORF1序列中的删除，表达截短的蛋白质，并检查其活动，以划定区域的ORF1p负责这些不同的功能。通过酵母双杂交分析和GST pull-down分析，蛋白质-蛋白质相互作用结构域被定义为一个卷曲螺旋结构域，它包含约三分之一的蛋白质在其N端附近。基于UV-交联，电泳迁移率变化测定和退火测定所获得的数据，ORF 1p的C-末端三分之一对于核酸结合和促进互补寡核苷酸的退火都是必要的和足够的。将这些活动分离到ORF1p的不同结构域中，将有助于对该蛋白的结构和功能进行详细的生化分析，并了解其在L1反转录转座过程中的作用。(C)北京大学出版社.
LINE-1, or L1, is a non-LTR retrotransposon in mammals. Retrotransposition of L1 requires the action of two element-encoded proteins, ORF1p and ORF2p. ORF2p provides essential enzymatic activities for the reverse transcription and integration of a newly transposed copy of L1, whereas the exact role of ORF1p is less well understood. The 43 kDa ORF1p copurifies as a large complex with L1 RNA in extracts of human and mouse cells. Mouse ORF1p purified from Escherichia coli binds RNA and single-stranded DNA in vitro, exhibits nucleic acid chaperone activity, and is capable of protein-protein interaction. Ln this study we create a series of deletions in the ORF1 sequence, express the truncated proteins and examine their activities to delineate the region of ORF1p responsible for these different functions. By both yeast two-hybrid analysis and GST pull-down assay, the protein-protein interaction domain is defined as a coiled-coil domain that encompasses about one third of the protein near its N terminus. Based on data obtained with UV-cross-linking, electrophoretic mobility-shift assay and an annealing assay, the C-terminal one third of ORF1p is both necessary and sufficient for nucleic acid binding and to promote annealing of complementary oligonucleotides. Separation of these activities into different domains of ORF1p will facilitate detailed biochemical analyses of the structure and function of this protein and understanding of its role during L1 retrotransposition. (C) 2000 Academic Press.