| Research Abstract |
The Sex-lethal (Sxl) protein of Drosophila melanogaster regulates alternative splicing of the transformer (tra) mRNA precursor by binding to the tra polypyrimidine tract in the sex determination cascade. The Sxl protein has two tandemly-linked RNA binding domains. As the amino-terminal RBD (RBD1) of the Sxl protein exhibits low sequence homology to the typical RBDs, paticularly at the putative functional residues, it was difficult to unambiguously locate the RNP1 and RNP2 motifs. Therefore, at first, we defined the amino and carboxy-terminal borders of the first RNA-binding domain (RBD1) of the Sxl protein by limited tryptic digestion. By replacement of Phe 166 by Tyr, we constructed a highly soluble mutant, which exhibits the same RNA-binding properties as those of the wild-type. Using this mutant protein, we performed NMR measurements, andelucidated the secondary and tertiary structures of the Sxl RBD1 in solution.
Second, we have determined the crystal structure of the complex between the two tandemly-arranged RNA binding domains of the Sxl protein and a 12-nucleotide, single-stranded RNa derived from the tra polypyrimidine tract. The two RNA-binding domains have their beta-sheet platforms facing each other, to form a V-shaped cleft. The RNA is characteristically extended and bound in this cleft, where the UGUUUUUUU sequence is specifically recognised by the protein. This result provides the first insight into the mechanism by which a protein binds specifically to a cognate RNA that has no intramolecular base pairs.
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