Interaction of N-terminal domain of U1A protein with an RNA stem/loop

Kathleen B. Hall, W. Tom Stump

Research output: Contribution to journalArticlepeer-review

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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-termlnal domain is unknown. Here, we describe experiments using a 102 amino acid N-termlnal RRM of U1A (102A) and a 25-nucleotide RNA stem/loop to measure the binding constants and thermodynamic parameters of this RNA:proteln complex. Using nitrocellulose filter binding, we measure a dissociation constant KD = 2×10-11 M in 250 mM NaCI, 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 (AG°) of this complex is about -14 kcal/mol in these conditions. Determination of the salt dependence of the binding suggests that at least 8 ionpairs are formed upon complex formation. A mutation in the RNA loop sequence reduces the affinity 10× , or about 10% of the total free energy.

Original languageEnglish
Pages (from-to)4283-4290
Number of pages8
JournalNucleic acids research
Issue number16
StatePublished - Aug 25 1992


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