A thermodynamic and mutational analysis of an RNA purine loop as a protein binding site.
The thermal stability and protein binding of a 36 nucleotide RNA hairpin containing an internal loop were studied under various solution conditions. Yeast ribosomal protein L32 binds to its transcript and small RNAs which reproduce the L32 transcript's secondary structure have been examined. Replacement of the internal loop with canonical base pairs did not affect the salt dependence of the melting temperature suggesting that both molecules adopt a linear shape. Several electrostatic contacts are formed on binding to a ribosomal fusion protein, but Mg+2 is not required for binding. The RNA protein complex is stable up to 50 degrees C. Two internal loop deletion mutants have similar thermodynamic stabilities and chemical and enzymatic reactivities, but fail to bind the fusion protein. However, several of the internal loop bases of the deletion mutants are moderately reactive to chemical agents whereas the wild type loop sequence displayed a mixed pattern of protection and hyperreactivity.
White
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Chemistry Department, Bryn Mawr College, PA 19010, USA.
Li
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Rauch
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ME
D001483:Base Sequence; D001665:Binding Sites; D002872:Chromosome Deletion; D018257:Helix-Loop-Helix Motifs; D016014:Linear Models; D008969:Molecular Sequence Data; D009154:Mutation; D009691:Nucleic Acid Denaturation; D011685:Purine Nucleotides / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D012492:Salts; D012996:Solutions; D013696:Temperature; D013816:Thermodynamics
D011685:Purine Nucleotides; D016601:RNA-Binding Proteins; D012492:Salts; D012996:Solutions