IDENTIFICATION AND MOLECULAR-CLONING OF A HUMAN SELENOCYSTEINE INSERTION SEQUENCE-BINDING PROTEIN - A BIFUNCTIONAL ROLE FOR DNA-BINDING PROTEIN-B

Prokaryotic and eukaryotic cells incorporate the unusual amino acid se lenocysteine at a UGA codon, which conventionally serves as a terminat ion signal, Translation of eukaryotic selenoprotein mRNA requires a nu cleotide selenocysteine insertion sequence in the 3'-untranslated regi on, We report the molecular cloning of the binding protein that recogn izes the selenocysteine insertion sequence element in human cellular g lutathione peroxidase gene (GPX1) transcripts and its identification a s DNA-binding protein B, a member of the EFIA/dbpB/YB-1 family. The pr edicted amino acid sequence contains four arginine-rich RNA-binding mo tifs, and one segment shows strong homology to the human immunodeficie ncy virus Tat domain. Recombinant DNA-binding protein B binds the sele nocysteine insertion sequence elements from the GPX1 and type I iodoth yronine 5'-deiodinase genes in RNA electrophoretic mobility shift assa ys and competes with endogenous GPX1 selenocysteine insertion sequence binding activity in COS-1 cytosol extracts. Addition of antibody to D NA-binding protein B to COS-1 electromobility shift assays produces a slowly migrating ''supershift'' band. The molecular cloning and identi fication of DNA-binding protein B as the first eukaryotic selenocystei ne insertion sequence-binding protein opens the way to the elucidation of the entire complex necessary for the alternative reading of the ge netic code that permits translation of selenoproteins.