Mammalian heterogeneous nuclear ribonucleoprotein A1. Nucleic acid binding properties of the COOH-terminal domain.
A1 is a core protein of the eukaryotic heterogeneous nuclear ribonucleoprotein complex and is under study here as a prototype single-stranded nucleic acid-binding protein. A1 is a two-domain protein, NH2-terminal and COOH-terminal, with highly conserved primary structure among vertebrate homologues sequenced to date. It is well documented that the NH2-terminal domain has single-stranded DNA and RNA binding activity. We prepared a proteolytic fragment of rat A1 representing the COOH-terminal one-third of the intact protein, the region previously termed COOH-terminal domain. This purified fragment of 133 amino acids binds to DNA and also binds tightly to the fluorescent reporter poly(ethenoadenylate), which is used to access binding parameters. In solution with 0.41 M NaCl, the equilibrium constant is similar to that observed with A1 itself, and binding is cooperative. The purified COOH-terminal fragment can be photochemically cross-linked to bound nucleic acid, confirming that COOH-terminal fragment residues are in close contact with the polynucleotide lattice. These binding results with isolated COOH-terminal fragment indicate that the COOH-terminal domain in intact A1 can contribute directly to binding properties. Contact between both COOH-terminal domain and NH2-terminal domain residues in an intact A1:poly(8-azidoadenylate) complex was confirmed by photochemical cross-linking.
Kumar
A
A
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Casas-Finet
J R
JR
Luneau
C J
CJ
Karpel
R L
RL
Merrill
B M
BM
Williams
K R
KR
Wilson
S H
SH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism*; D003432:Cross-Linking Reagents; D010450:Endopeptidases; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010446:Peptide Fragments / Q000302:isolation & purification; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear
D003432:Cross-Linking Reagents; D015345:Oligonucleotide Probes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D010450:Endopeptidases