Isolation and molecular characterization of a cDNA encoding the 23-kDa subunit of human RNA polymerase II

Abstract

We have shown that antibodies against native calf thymus RNA polymerase II and antibodies against its 23-kDa subunit cross-reacted with the 23-kDa subunit of human RNA polymerase II. Immunoglobin G (IgG) against the 23-kDa subunit of calf thymus RNA polymerase II inhibited transcription in vitro from the adenovirus major late promoter. By immunoscreening of a human placenta lambda gt11 cDNA library with IgG against native CT RNA polymerase II and with IgG against its 23-kDa subunit, we isolated and characterized a full length 1.2-kilobase cDNA. We also generated oligonucleotide probes from a sequence of amino acid residues obtained by a modified peptide microsequencing procedure. The cDNAs isolated both from oligoscreening and immunoscreening were identical. The amino acid sequence deduced from the nucleotide sequence analysis indicates a polypeptide of 197 amino acid (23 kDa). The in vitro translation product of human cDNA HP-23 was precipitated by IgG against the 23-kDa subunit of CT RNA polymerase II. The amino acid sequence deduced from HP-23 showed no obvious homology with Escherichia coli RNA polymerase subunits or with any of its sigma factors.