title: Assignment of the L30-mRNA complex using selective isotopic labeling and RNA mutants.
abstract: The helix-loop-helix structure formed in the pre-mRNA and the mRNA of L30, a ribosomal protein from the yeast Saccharomyces cerevisiae, serves as an auto-regulatory binding site for the protein to suppress the L30 synthesis upon overproduction. Using a 33-nucleotide model RNA, the structures of the L30 binding site RNA in the presence and absence of the protein were investigated using nuclear magnetic resonance (NMR) spectroscopy. Homonuclear and(13)C/(15)N-based resonance assignments and spectral comparisons indicated that the purine-rich internal loop is dynamic in the free RNA but becomes ordered in the presence of L30 protein. Although the resonances in the loop region are sharper and more disperse in the bound RNA, their assignment was extremely challenging, due to spectral complexity and broadened resonances caused by local dynamics. Two strategies, namely selective(13)C/(15)N-labeling and NMR analyses of five complexes with RNA mutants, were used to overcome these difficulties. Only using these approaches could assignment of the internal loop resonances and identification of the unusual NOEs and nucleotide conformations within the internal loop be made. In the case of structural determination of the L30-mRNA complex, it was critical to be able to take advantage of the available biochemical information in order to complete the structure determination.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: H
authors.initials: H
authors.lastname: Mao
authors.forename: J R
authors.initials: JR
authors.lastname: Williamson
mesh_terms: D001483:Base Sequence; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics
chemical_list: D012333:RNA, Messenger; D012269:Ribosomal Proteins; C035201:ribosomal protein L30