title: Native Top-Down Mass Spectrometry of TAR RNA in Complexes with a Wild-Type tat Peptide for Binding Site Mapping.
abstract: Ribonucleic acids (RNA) frequently associate with proteins in many biological processes to form more or less stable complex structures. The characterization of RNA-protein complex structures and binding interfaces by nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography, or strategies based on chemical crosslinking, however, can be quite challenging. Herein, we have explored the use of an alternative method, native top-down mass spectrometry (MS), for probing of complex stoichiometry and protein binding sites at the single-residue level of RNA. Our data show that the electrostatic interactions between HIV-1 TAR RNA and a peptide comprising the arginine-rich binding region of tat protein are sufficiently strong in the gas phase to survive phosphodiester backbone cleavage of RNA by collisionally activated dissociation (CAD), thus allowing its use for probing tat binding sites in TAR RNA by top-down MS. Moreover, the MS data reveal time-dependent 1:2 and 1:1 stoichiometries of the TAR-tat complexes and suggest structural rearrangements of TAR RNA induced by binding of tat peptide.
authors.affiliation: Institut für Organische Chemie and Center for Molecular Biosciences Innsbruck (CMBI), Universität Innsbruck, Innrain 80-82, 6020, Innsbruck, Austria.
authors.forename: Eva-Maria
authors.identifier: 0000-0002-9519-3110
authors.initials: EM
authors.lastname: Schneeberger
authors.affiliation: Institut für Organische Chemie and Center for Molecular Biosciences Innsbruck (CMBI), Universität Innsbruck, Innrain 80-82, 6020, Innsbruck, Austria.
authors.forename: Kathrin
authors.identifier: 0000-0002-4978-0883
authors.initials: K
authors.lastname: Breuker
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D015696:Gene Products, tat / Q000737:chemistry / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D015696:Gene Products, tat; D012367:RNA, Viral
keywords: RNA; binding sites; collisionally activated dissociation; mass spectrometry; proteins