Interaction of N-terminal domain of U1A protein with an RNA stem/loop.
The U1A protein is a sequence-specific RNA binding protein found in the U1 snRNP particle where it binds to stem/loop II of U1 snRNA. U1A contains two 'RNP' or 'RRM' (RNA Recognition Motif) domains, which are common to many RNA-binding proteins. The N-terminal RRM has been shown to bind specifically to the U1 RNA stem/loop, while the RNA target of the C-terminal domain is unknown. Here, we describe experiments using a 102 amino acid N-terminal RRM of U1A (102A) and a 25-nucleotide RNA stem/loop to measure the binding constants and thermodynamic parameters of this RNA:protein complex. Using nitrocellulose filter binding, we measure a dissociation constant KD = 2 x 10(-11) M in 250 mM NaCl, 2 mM MgC2, and 10 mM sodium cacodylate, pH 6 at room temperature, and a half-life for the complex of 5 minutes. The free energy of association (delta G degrees) of this complex is about -14 kcal/mol in these conditions. Determination of the salt dependence of the binding suggests that at least 8 ion-pairs are formed upon complex formation. A mutation in the RNA loop sequence reduces the affinity 10 x, or about 10% of the total free energy.
Hall
K B
KB
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO 63110.
Stump
W T
WT
D001483:Base Sequence; D003001:Cloning, Molecular; D004586:Electrophoresis; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; D012261:Ribonucleoproteins / Q000378:metabolism*; D012492:Salts / Q000378:metabolism
D010446:Peptide Fragments; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; D012261:Ribonucleoproteins; D012492:Salts; C075339:U1A protein