title: Identification and solution structure of a highly conserved C-terminal domain within ORF1p required for retrotransposition of long interspersed nuclear element-1.
abstract: Long interspersed nuclear element-1 (LINE-1 or L1) retrotransposons comprise a large fraction of the human and mouse genomes. The mobility of these successful elements requires the protein encoded by open reading frame-1 (ORF1p), which binds single-stranded RNA with high affinity and functions as a nucleic acid chaperone. In this report, we have used limited proteolysis, filter binding, and NMR spectroscopy to characterize the global structure of ORF1p and the three-dimensional structure of a highly conserved RNA binding domain. ORF1p contains three structured regions, a coiled-coil domain, a middle domain of unknown function, and a C-terminal domain (CTD). We show that high affinity RNA binding by ORF1p requires the CTD and residues within an amino acid protease-sensitive segment that joins the CTD to the middle domain. Insights in the mechanism of RNA binding were obtained by determining the solution structure of the CTD, which is shown to adopt a novel fold consisting of a three-stranded beta sheet that is packed against three alpha-helices. An RNA binding surface on the CTD has been localized using chemical shift perturbation experiments and is proximal to residues previously shown to be essential for retrotransposition, RNA binding, and chaperone activity. A similar structure and mechanism of RNA binding is expected for all vertebrate long interspersed nuclear element-1 elements, since residues encoding the middle, protease-sensitive segment, and CTD are highly conserved.
authors.affiliation: Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095, USA.
authors.forename: Kurt
authors.initials: K
authors.lastname: Januszyk
authors.forename: Patrick Wai-Lun
authors.initials: PW
authors.lastname: Li
authors.forename: Valerie
authors.initials: V
authors.lastname: Villareal
authors.forename: Dan
authors.initials: D
authors.lastname: Branciforte
authors.forename: Haihong
authors.initials: H
authors.lastname: Wu
authors.forename: Yongming
authors.initials: Y
authors.lastname: Xie
authors.forename: Juli
authors.initials: J
authors.lastname: Feigon
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Loo
authors.forename: Sandra L
authors.initials: SL
authors.lastname: Martin
authors.forename: Robert T
authors.initials: RT
authors.lastname: Clubb
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D007700:Kinetics; D020084:Long Interspersed Nucleotide Elements / Q000235:genetics*; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D018626:Retroelements / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D018626:Retroelements