Spatial assembly and RNA binding stoichiometry of a LINE-1 protein essential for retrotransposition.
LINE-1, or L1, is a highly successful retrotransposon in mammals, comprising 17% and 19% of the human and mouse genomes, respectively. L1 retrotransposition and hence amplification requires the protein products of its two open reading frames, ORF1 and ORF2. The sequence of the ORF1 protein (ORF1p) is not related to any protein with known function. ORF1p has RNA binding and nucleic acid chaperone activities that are both required for retrotransposition. Earlier studies have shown that ORF1p forms a homotrimer with an asymmetric dumbbell shape, in which a rod separates a large end from a small end. Here, we determine the topological arrangement of monomers within the homotrimer by comparing atomic force microscopy (AFM) images of the full ORF1p with those of truncations containing just the N or C-terminal regions. In addition, AFM images of ORF1p bound to RNA at high protein/RNA molar ratios show that ORF1p can form tightly packed clusters on RNA, with binding occurring at the C-terminal domain. The number of bound ORF1p trimers increases with increasing length of the RNA, revealing that the binding site size is about 50 nt, a value confirmed by nitrocellulose filter binding under stoichiometric conditions. These results are consistent with a role for ORF1p during L1 retrotransposition that includes both coating the RNA and acting as a nucleic acid chaperone. Furthermore, these in vitro L1 ribonucleoprotein particles provide insight into the structure of the L1 retrotransposition intermediate.
Basame
Solomon
S
Molecular Machines Laboratory, Department of Chemistry, University of New Mexico, Albuquerque, NM 87131, USA.
Wai-lun Li
Patrick
P
Howard
Grant
G
Branciforte
Dan
D
Keller
David
D
Martin
Sandra L
SL
D000818:Animals; D006801:Humans; D020084:Long Interspersed Nucleotide Elements; D051379:Mice; D018625:Microscopy, Atomic Force; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames; D011485:Protein Binding; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism*; D018626:Retroelements
D018626:Retroelements; D012313:RNA