CHEMICAL-SHIFT MAPPING OF THE RNA-BINDING INTERFACE OF THE MULTIPLE-RBD PROTEIN SEX-LETHAL

The Drosophila protein Sex-lethal (Sx1) contains two RNP consensus-typ e RNA-binding domains (RBDs) separated by a short linker sequence. Bot h domains are essential for high-affinity binding to the single-strand ed polypyrimidine tract (PPT) within the regulated 3' splice site of t he transformer (tra) pre-mRNA. In this paper, the effect of RNA bindin g to a protein fragment containing both RBDs from Sx1 (Sx1-RBD1+2) has been characterized by heteronuclear NMR. Nearly complete (85-90%) bac kbone resonance assignments have been obtained for unbound and RNA-bou nd states of Sx1-RBD1+2. A comparison of amide H-1 and N-15 chemical s hifts between free and bound states has highlighted residues which res pond to RNA binding. The beta-sheets in both RBDs (RBD1 and RBD2) form an RNA interaction surface, as has been observed in other RBDs. A sig nificant number of residues display different behavior when comparing RBD1 and RBD2. This argues for a model in which RBD1 and RBD2 of Sx1 h ave different or nonanalogous points of interaction with the tra PPT. R-142 (in RBD2) exhibits the largest chemical shift change upon RNA bi nding. The role of R-142 in RNA binding was tested by measuring the K- d of a mutant of Sx1-RBD1+2 in which R-142 was replaced by alanine. Th is mutant lost the ability to bind RNA, showing a correlation with the chemical. shift difference data. The RNA-binding affinities of two ot her mutants, F(146)A and (TI)-I-138, were also shown to correlate with the NMR observations.