title: RNA recognition by the DNA end-binding Ku heterodimer.
abstract: Most nucleic acid-binding proteins selectively bind either DNA or RNA, but not both nucleic acids. The Saccharomyces cerevisiae Ku heterodimer is unusual in that it has two very different biologically relevant binding modes: (1) Ku is a sequence-nonspecific double-stranded DNA end-binding protein with prominent roles in nonhomologous end-joining and telomeric capping, and (2) Ku associates with a specific stem-loop of TLC1, the RNA subunit of budding yeast telomerase, and is necessary for proper nuclear localization of this ribonucleoprotein enzyme. TLC1 RNA-binding and dsDNA-binding are mutually exclusive, so they may be mediated by the same site on Ku. Although dsDNA binding by Ku is well studied, much less is known about what features of an RNA hairpin enable specific recognition by Ku. To address this question, we localized the Ku-binding site of the TLC1 hairpin with single-nucleotide resolution using phosphorothioate footprinting, used chemical modification to identify an unpredicted motif within the hairpin secondary structure, and carried out mutagenesis of the stem-loop to ascertain the critical elements within the RNA that permit Ku binding. Finally, we provide evidence that the Ku-binding site is present in additional budding yeast telomerase RNAs and discuss the possibility that RNA binding is a conserved function of the Ku heterodimer.
authors.affiliation: Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado BioFrontiers Institute, Boulder, Colorado 80309-0596, USA.
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Dalby
authors.forename: Karen J
authors.initials: KJ
authors.lastname: Goodrich
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Pfingsten
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Cech
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D003028:CME-Carbodiimide / Q000031:analogs & derivatives / Q000737:chemistry; D002467:Cell Nucleus / Q000737:chemistry / Q000235:genetics; D018983:DNA Footprinting / Q000379:methods; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D055029:Inverted Repeat Sequences; D009154:Mutation; D009690:Nucleic Acid Conformation; D059372:Nucleotide Motifs; D054735:Phosphorothioate Oligonucleotides / Q000737:chemistry; D025941:Protein Interaction Mapping; D012313:RNA / Q000235:genetics / Q000378:metabolism; D059367:RNA Cleavage; D012331:RNA, Fungal / Q000737:chemistry* / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics; D013463:Sulfuric Acid Esters / Q000737:chemistry; D019098:Telomerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D054735:Phosphorothioate Oligonucleotides; D012331:RNA, Fungal; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D013463:Sulfuric Acid Esters; C081647:high affinity DNA-binding factor, S cerevisiae; C422952:telomerase RNA; C060331:1-cyclohexyl-3-(2-(4-morpholinyl)ethyl)carbodiimide; D003028:CME-Carbodiimide; D012313:RNA; D019098:Telomerase; C007482:dimethyl sulfate
keywords: Ku heterodimer; RNA binding; chemical modification; footprinting; telomerase