Cloning of Two Proximal Sequence Element-Binding Transcription Factor Subunits (γ and δ) That Are Required for Transcription of Small Nuclear RNA Genes by RNA Polymerases II and III and Interact with the TATA-Binding Protein

Abstract

The proximal sequence element (PSE)-binding transcription factor (PTF) specifically recognizes the PSEs of both RNA polymerase II- and RNA polymerase III-transcribed small nuclear RNA (snRNA) genes. We previously have shown that PTF purified from human HeLa cells is a multisubunit complex of four polypeptides designated PTF α, -β, -γ, and -δ. We now report the isolation and expression of cDNAs encoding PTF γ and PTF δ, as well as functional studies with cognate antibodies that recognize the native PTF complex in HeLa extracts. Immunoprecipitation studies confirm that the four PTF subunits originally found to copurify during conventional chromatography indeed form a tightly associated complex; they further show that the PTF so defined, including the γ and δ subunits specifically, is essential for transcription of both class II and class III snRNA genes. Immunoprecipitation assays also show a weak substoichiometric association of the TATA-binding protein (TBP) with PTF, consistent with the previous report of a PTF-related complex (SNAPc) containing substoichiometric levels of TBP and a component (SNAPc43) identical in sequence to the PTF γ reported here. Glutathione S-transferase pulldown assays further indicate relatively strong direct interactions of both recombinant PTF γ and PTF δ with TBP, consistent either with the natural association of TBP with PTF in a semistable TBP-TBP-associated factor complex or with possible functional interactions between PSE-bound PTF and TATA-bound TBP during promoter activation. In addition, we show that in extracts depleted of TBP and TBP-associated factors, transcription from the U1 promoter is restored by recombinant TBP but not by TFIID or TFIIIB, indicating that transcription of class II snRNA genes requires a TBP complex different from the one used for mRNA-encoding genes.