Physical and immunological characterization of human transcription factor IIIA

Abstract

Human transcription factor IIIA (htFIIIA), specifically required for transcription of the gene for 5S ribosomal RNA has been characterized with respect to some of its physical, immunological and functional properties. TFIIIA from HeLa cells, which selectively binds 5S RNA, is a monomer of approximately 35 kDa with a Stokes' radius of approximately 2.65 nm and a sedimentation coefficient of approximately 2.8 S. These values indicate that the human protein is of rather globular shape and hence diverges not only in molecular mass but also in most of the molecular properties from its highly asymmetric counterpart in Xenopus laevis oocytes. By raising specific polyclonal antibodies against hTFIIIA it was shown in Western immunoblots that there was no cross-reaction between anti-hTFIIIA antibodies and the amphibian protein. Conversely, monoclonal antibodies against three domains of X. laevis TFIIIA antibodies and the amphibian protein. Conversely, monoclonal antibodies against three domains of X. laevis TFIIIA did not cross-react with the human transcription factor. The polyclonal antisera raised against hTFIIIA specifically neutralized binding of the human transcription factor to 5S DNA and abolished in vitro transcription of 5S RNA but these antibodies were unable to inhibit 5S RNA synthesis in cellular extracts from Xenopus, Drosophila or yeast cells. Finally, the species variation of TFIIIA could be substantiated by electrophoretic mobility shift assays revealing preferential binding of hTFIIIA to the homologous 5S RNA gene.