Deletion analysis defines distinct functional domains for protein-protein and nucleic acid interactions in the ORF1 protein of mouse LINE-1.
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. In 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.
Martin
S L
SL
Department of Cellular and Structural Biology, University of Colorado School of Medicine, Denver, CO 80262, USA. sandy.martin@uchsc.edu
Li
J
J
Weisz
J A
JA
D000818:Animals; D020029:Base Pairing; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020084:Long Interspersed Nucleotide Elements / Q000235:genetics*; D051379:Mice; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D017384:Sequence Deletion / Q000235:genetics*; D013329:Structure-Activity Relationship; D020798:Two-Hybrid System Techniques; D014466:Ultraviolet Rays
D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D010446:Peptide Fragments; D016601:RNA-Binding Proteins; D004247:DNA