title: The L7Ae protein binds to two kink-turns in the Pyrococcus furiosus RNase P RNA.
abstract: The RNA-binding protein L7Ae, known for its role in translation (as part of ribosomes) and RNA modification (as part of sn/oRNPs), has also been identified as a subunit of archaeal RNase P, a ribonucleoprotein complex that employs an RNA catalyst for the Mg(2+)-dependent 5' maturation of tRNAs. To better understand the assembly and catalysis of archaeal RNase P, we used a site-specific hydroxyl radical-mediated footprinting strategy to pinpoint the binding sites of Pyrococcus furiosus (Pfu) L7Ae on its cognate RNase P RNA (RPR). L7Ae derivatives with single-Cys substitutions at residues in the predicted RNA-binding interface (K42C/C71V, R46C/C71V, V95C/C71V) were modified with an iron complex of EDTA-2-aminoethyl 2-pyridyl disulfide. Upon addition of hydrogen peroxide and ascorbate, these L7Ae-tethered nucleases were expected to cleave the RPR at nucleotides proximal to the EDTA-Fe-modified residues. Indeed, footprinting experiments with an enzyme assembled with the Pfu RPR and five protein cofactors (POP5, RPP21, RPP29, RPP30 and L7Ae-EDTA-Fe) revealed specific RNA cleavages, localizing the binding sites of L7Ae to the RPR's catalytic and specificity domains. These results support the presence of two kink-turns, the structural motifs recognized by L7Ae, in distinct functional domains of the RPR and suggest testable mechanisms by which L7Ae contributes to RNase P catalysis.
authors.affiliation: Department of Chemistry & Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Stella M
authors.initials: SM
authors.lastname: Lai
authors.affiliation: Department of Chemistry & Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Lien B
authors.initials: LB
authors.lastname: Lai
authors.affiliation: Department of Chemistry & Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA gopalan.5@osu.edu.
authors.forename: Mark P
authors.initials: MP
authors.lastname: Foster
authors.affiliation: Department of Chemistry & Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA gopalan.5@osu.edu.
authors.forename: Venkat
authors.initials: V
authors.lastname: Gopalan
mesh_terms: D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D009690:Nucleic Acid Conformation; D017433:Protein Structure, Secondary; D020027:Pyrococcus furiosus / Q000235:genetics; D019642:RNA, Archaeal / Q000737:chemistry* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D043262:Ribonuclease P / Q000737:chemistry* / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D019642:RNA, Archaeal; D016601:RNA-Binding Proteins; D043262:Ribonuclease P