TY - JOUR
T1 - Interaction of N-terminal domain of U1A protein with an RNA stem/loop
AU - Hall, Kathleen B.
AU - Stump, W. Tom
N1 - Funding Information:
We thank members of Prof. Tim Lohman's and Prof. Gary Acker's laboratories for their interest and critical comments, and Dr. Isaac Wong for his help with Kaleidograph. This work was supported by the Lucille P. Markey Charitable Trust (# 90-7) (KBH).
PY - 1992/8/25
Y1 - 1992/8/25
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0026713783&partnerID=8YFLogxK
U2 - 10.1093/nar/20.16.4283
DO - 10.1093/nar/20.16.4283
M3 - Article
C2 - 1508720
AN - SCOPUS:0026713783
VL - 20
SP - 4283
EP - 4290
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 16
ER -