Mammalian heterogeneous nuclear ribonucleoprotein complex protein A1. Large-scale overproduction in Escherichia coli and cooperative binding to single-stranded nucleic acids

• Published in: The Journal of biological chemistry 1988-01, Vol.263 (2), p.1063-1071
• Main Authors: Cobianchi, F, Karpel, R L, Williams, K R, Notario, V, Wilson, S H
• Format: Article
• Language: English
• Subjects: Amino Acids - analysis; Analytical, structural and metabolic biochemistry; Base Sequence; Binding and carrier proteins; Biological and medical sciences; Biotechnology; Cellulose - analogs & derivatives; Cellulose - metabolism; DNA - analogs & derivatives; DNA - analysis; DNA - metabolism; DNA, Single-Stranded - metabolism; Escherichia coli; Escherichia coli - metabolism; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Genetic engineering; Genetic technics; Heterogeneous Nuclear Ribonucleoprotein A1; Heterogeneous-Nuclear Ribonucleoprotein Group A-B; Heterogeneous-Nuclear Ribonucleoproteins; Methods. Procedures. Technologies; Molecular cloning; Molecular Sequence Data; Poly A; Proteins; Recombinant Proteins - metabolism; Ribonucleoproteins - biosynthesis; Ribonucleoproteins - metabolism; RNA - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)35461-4

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.