Biochemical characterization of the Oct-2 POU domain with implications for bipartite DNA recognition

• Published in: Biochemistry (Easton) Vol. 31; no. 25; pp. 5841 - 5848
• Main Authors: Botfield, Martyn C, Jancso, Agnes, Weiss, Michael A
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 30.06.1992
• Subjects: Amino Acid Sequence; B-Lymphocytes - chemistry; Base Sequence; Binding Sites; Biological and medical sciences; Chromatography, High Pressure Liquid; DNA - chemistry; DNA - metabolism; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Drug Stability; Enhancer Elements, Genetic; Escherichia coli - genetics; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Gene Expression; Humans; Magnetic Resonance Spectroscopy; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Mutagenesis; Octamer Transcription Factor-2; Peptide Fragments - metabolism; Plasmids; Promoter Regions, Genetic; Protein Conformation; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Temperature; Thermolysin - metabolism; Transcription Factors; Transcription. Transcription factor. Splicing. Rna processing; Site specificity; Binding capacity; Structural unit; DNA; Mechanism; Recognition
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00140a020

B-cell specific regulation of immunoglobulin gene expression provides a model for the interaction of promoter and enhancer elements with eukaryotic sequence-specific DNA binding proteins. A critical element of this system, the octamer site (5'-ATGCAAAT-3'), is recognized by the B-cell transcription factor Oct-2. Octamer recognition is mediated by the POU domain, a conserved structural motif which--like the zinc finger and leucine zipper--defines a family of related transcription factors. Homologies among POU sequences suggest a bipartite structure, consisting of an N-terminal POU-specific subdomain and C-terminal variant homeodomain connected by a linker of variable length and sequence. As a first step toward a molecular understanding of the Oct-2 POU domain and its mechanism of DNA recognition, we have overexpressed in Escherichia coli the intact POU domain and subdomains as thrombin-cleavable fusion proteins and have purified these fragments to homogeneity following digestion with thrombin. Biochemical and biophysical characterization yields the following results. (i) The intact POU domain (166 residues) is monomeric and exhibits high-affinity octamer-specific DNA-binding activity. (ii) Limited proteolytic digestion demonstrates that the POU domain contains two proteolytically stable subdomains (the POU-specific subdomain and the variant homeodomain) connected by a proteolytically sensitive linker. (iii) The isolated subdomains are each monomeric and do not interact to form noncovalent heterodimers.