Cloning and Characterization of the β Subunit of Human Proximal Sequence Element-Binding Transcription Factor and Its Involvement in Transcription of Small Nuclear RNA Genes by RNA Polymerases II and III

Abstract

The proximal sequence element (PSE)-binding transcription factor (PTF), which binds the PSE of both RNA polymerase II- and RNA polymerase III-transcribed mammalian small nuclear RNA (snRNA) genes, is essential for their transcription. We previously reported the purification of human PTF, a complex of four subunits, and the molecular cloning and characterization of PTF γ and δ subunits. Here we describe the isolation and expression of a cDNA encoding PTFβ, as well as functional studies using anti-PTFβ antibodies. Native PTFβ, in either protein fractions or a PTF–Oct-1–DNA complex, can be recognized by polyclonal antibodies raised against recombinant PTFβ. Immunodepletion studies show that PTFβ is required for transcription of both classes of snRNA genes in vitro. In addition, immunoprecipitation analyses demonstrate that substantial and similar molar amounts of TATA-binding protein (TBP) and TFIIIB90 can weakly associate with PTF at low salt conditions, but this association is dramatically reduced at high salt concentrations. Along with our previous demonstration of both physical interactions between PTFγ/PTFδ and TBP and the involvement of TFIIIB90 in the transcription of class III snRNA genes, these results are consistent with the notion that a TBP-containing complex related to TFIIIB is required for the transcription of class III snRNA genes, and acts through weak interaction with the four-subunit PTF.