NMR is being used to gather structural information on an RNA-binding protein complexed with its target RNA sequence. Sex determination in Drosophila results from a cascade of RNA-splicing regulated events which are triggered by the presence of a master switch protein, Sex-lethal. Sex-lethal binds a specific, uridine rich 3?-splice site on the messenger RNA of the transformer gene. Sex-lethal has been shown to contain two RNA-binding domains (RBDs) of the RNP consensus type. Our previous work has concentrated on the C-terminal RBD (Sxl-RBD2), where nearly all 1H, 13C, and 15N resonances were assigned and a family of structures were calculated to intermediate resolution. We are presently interested in obtaining structural information on the high-affininity complex of both RBDs (contained in a single polypeptide chain, RBD1+2) with the RNA 10-mer, 5?-GUUUUUUUUC-3?. First, we plan on obtaining sequence-specific resonance assignments of the protein backbone of RBD1+2 when complexed with the 10- mer. We are approaching this problem by collecting and analyzing 3D HNCO, HNCA, HN(CO)CA, HCACO, CBCA(CO)NH, 15N-separated TOCSY, and 15N-separated NOESY. It is hoped that we will obtain a complete map of the secondary structure of this protein in the bound state. We also hope to obtain rudimentary information on the solution motional dynamics of backbone amide NH vectors in RBD1+2 in the bound state. Therefore, we will collect 15N T2 data as is commonly done in the form of a 2D HSQC series. If time permits, we will also collect 15N T1 data.
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