Mammalian heterogeneous nuclear ribonucleoprotein complex protein A1. Large-scale overproduction in Escherichia coli and cooperative binding to single-stranded nucleic acids.
Characterization of mammalian heterogeneous nuclear ribonucleoprotein complex protein A1 is reported after large-scale overproduction of the protein in Escherichia coli and purification to homogeneity. A1 is a single-stranded nucleic acid binding protein of 320 amino acids and 34,214 Da. The protein has two domains. The NH2-terminal domain is globular, whereas the COOH-terminal domain of about 120 amino acids has low probability of alpha-helix structure and is glycinerich. Nucleic acid binding properties of recombinant A1 were compared with those of recombinant and natural proteins corresponding to the NH2-terminal domain. A1 bound to single-stranded DNA-cellulose with higher affinity than the NH2-terminal domain peptides. Protein-induced fluorescence enhancement was used to measure equilibrium binding properties of the proteins. A1 binding to poly (ethenoadenylate) was cooperative with the intrinsic association constant of 1.5 X 10(5) M-1 at 0.4 M NaCl and a cooperativity parameter of 30. The NH2-terminal domain peptides bound noncooperatively and with a much lower association constant. With these peptides and with intact A1, binding was fully reversed by increasing [NaCl]; yet. A1 binding was much less salt-sensitive than binding by the NH2-terminal domain peptides. A synthetic polypeptide analog of the COOH-terminal domain was prepared and was found to bind tightly to poly-(ethenoadenylate). The results are consistent with the idea that the COOH-terminal domain contributes to A1 binding through both cooperative protein-protein interaction and direct interaction with the nucleic acid.
D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D002482:Cellulose / Q000031:analogs & derivatives / Q000378:metabolism; D004247:DNA / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005456:Fluorescent Dyes; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D008969:Molecular Sequence Data; D011061:Poly A; D012313:RNA / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012261:Ribonucleoproteins / Q000096:biosynthesis* / Q000378:metabolism
D000596:Amino Acids; D004277:DNA, Single-Stranded; C033190:DNA-cellulose; D005456:Fluorescent Dyes; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D011061:Poly A; D012313:RNA; D002482:Cellulose; D004247:DNA