Chemical shift mapping of the RNA-binding interface of the multiple-RBD protein sex-lethal.
The Drosophila protein Sex-lethal (Sxl) contains two RNP consensus-type RNA-binding domains (RBDs) separated by a short linker sequence. Both domains are essential for high-affinity binding to the single-stranded polypyrimidine tract (PPT) within the regulated 3' splice site of the transformer (tra) pre-mRNA. In this paper, the effect of RNA binding to a protein fragment containing both RBDs from Sxl (Sxl-RBD1 + 2) has been characterized by heteronuclear NMR. Nearly complete (85-90%) backbone resonance assignments have been obtained for unbound and RNA-bound states of Sxl-RBD1 + 2. A comparison of amide 1H and 15N chemical shifts between free and bound states has highlighted residues which respond 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 significant number of residues display different behavior when comparing RBD1 and RBD2. This argues for a model in which RBD1 and RBD2 of Sxl have different or nonanalogous points of interaction with the tra PPT. R142 (in RBD2) exhibits the largest chemical shift change upon RNA binding. The role of R142 in RNA binding was tested by measuring the Kd of a mutant of Sxl-RBD1 + 2 in which R142 was replaced by alanine. This mutant lost the ability to bind RNA, showing a correlation with the chemical shift difference data. The RNA-binding affinities of two other mutants, F146A and T138I, were also shown to correlate with the NMR observations.
Lee
A L
AL
Department of Chemistry, University of California at Berkeley 94720-3204, USA.
Volkman
B F
BF
Robertson
S A
SA
Rudner
D Z
DZ
Barbash
D A
DA
Cline
T W
TW
Kanaar
R
R
Rio
D C
DC
Wemmer
D E
DE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003198:Computer Simulation; D029721:Drosophila Proteins; D004331:Drosophila melanogaster; D007301:Insect Hormones / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D012322:RNA Precursors / Q000737:chemistry* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D029721:Drosophila Proteins; D007301:Insect Hormones; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C067777:Sxl protein, Drosophila