Yeast ribosomal protein L32 recognizes an RNA G:U juxtaposition.
Yeast ribosomal protein L32, RPL32, specifically represses splicing by binding to a purine-rich asymmetric loop adjacent to the 5' splice site of its own transcript. A potential G:U pair closes the internal loop and the goal of the present study is to understand what features of the putative G:U pair are recognized by RPL32. Two RNA oligomers containing 10 and 13 nt were annealed to form a bimolecular stem-loop-stem protein-binding site. Protein binding to each of 16 sequence variants was examined using electrophoretic bandshift and filter-binding experiments. The proteins binds to only the duplex RNA and not to the individual oligomers, and the G:U pair is critical for full-strength binding. Mutational studies show that the duplex having a G:U has the highest protein affinity (Kd = 10 nM), followed by RNAs bearing G:A, C:C, U:A, U:C, or G:G. Duplexes containing the other possible pairs bind very weakly and Watson-Crick pairing does not favor protein binding. The G of the G:U is required for strong protein binding, but replacement by inosine reduces binding only modestly. Therefore, the minor groove guanine amino group is not a key protein recognition element. Both nucleotides of the pair influence the binding strength, but their contributions are in general not additive. These data imply that the G:U is probably paired and influences binding indirectly through its effect on the conformation of the RNA.
White
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Department of Chemistry, Bryn Mawr College, Pennsylvania 19010, USA swhite@brynmawr.edu.
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D001482:Base Composition; D001483:Base Sequence; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012326:RNA Splicing / Q000502:physiology; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism*
D012330:RNA, Double-Stranded; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; C041806:ribosomal protein L32