The proximal sequence element (PSE), found in both RNA polymerase II (Pol II)-and RNA Pol IIItranscribed small nuclear RNA (snRNA) genes, is specifically bound by the PSE-binding transcription factor (PTF). We have purified PTF to near homogeneity from HeLa cell extracts by using a combination of conventional and affinity chromatographic methods. Purified PTF is composed of four polypeptides with apparent molecular masses of 180, 55, 45, and 44 kDa. A combination of preparative electrophoretic mobility shift and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses has conclusively identified these four polypeptides as subunits of human PTF, while UV cross-linking experiments demonstrate that the largest subunit of PTF is in close contact with the PSE. The purified PTF activates transcription from promoters of both Pol II-and Pol III-transcribed snRNA genes in a PSE-dependent manner. In addition, we have investigated factor requirements in transcription of Pol III-dependent snRNA genes. We show that in extracts that have been depleted of TATA-binding protein (TBP) and associated factors, recombinant TBP restores transcription from U6 and 7SK promoters but not from the VAI promoter, whereas the highly purified TBP-TBPassociated factor complex TFIIIB restores transcription from the VAI but not the U6 or 7SK promoter. Furthermore, by complementation of heat-treated extracts lacking TFIIIC activity, we show that TFIIIC1 is required for transcription of both the 7SK and VAI genes, whereas TFIIIC2 is required only for transcription of the VAI gene. From these observations, we conclude (i) that PTF and TFIIIC2 function as gene-specific factors for PSE-and B-box-containing Pol III genes, respectively, (ii) that the form of TBP used by class III genes with upstream promoter elements differs from the form used by class III genes with internal promoters, and (iii) that TFIIIC1 is required for both internal and external Pol III promoters.Mammalian small nuclear RNA (snRNA) genes contain related promoter structures, but some (class II) are transcribed by RNA polymerase II (Pol II), whereas others (class III) are transcribed by RNA Pol III (reviewed in references 8, 15, 23, 30, and 37). Class II snRNA genes (e.g., U1 to U5) contain two regulatory elements in the 5Ј-flanking region: a distal sequence element (DSE) located approximately 220 bp upstream of the transcription start site, and a proximal sequence element (PSE) centered around Ϫ55. The DSE contains at least one copy of the octamer motif and functions as an upstream enhancer, whereas the PSE is an essential promoter element which functions in start site selection and may be required for accurate 3Ј-end formation (17,33,35). The promoters of class III snRNA genes (e.g., 7SK and U6) are located solely in the 5Ј-flanking region and lack intragenic control elements typical of most other class III genes (9, 29). Like their class II snRNA gene counterparts, class III snRNA genes have similar DSE and PSE configurations but additionally contain a TATA-like sequen...

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 gamma and delta subunits. Here we describe the isolation and expression of a cDNA encoding PTF beta, as well as functional studies using anti-PTF beta antibodies. Native PTF beta, in either protein fractions or a PTF-Oct-1-DNA complex, can be recognized by polyclonal antibodies raised against recombinant PTF beta. Immunodepletion studies show that PTF beta 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 PTF gamma/PTF delta 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.

contributed equally to this workWe have shown previously that the TFIIIC1/TFIIIC1¢ fraction interacts speci®cally with the VA1 terminator regions to affect both termination and initiation/reinitiation of transcription by human RNA polymerase III. Here, we further puri®ed the VA1 terminatorbinding factor to apparent homogeneity and found, by peptide sequence analysis, that it belongs to the NF1 protein family. NF1 interacts speci®cally with the NF1-binding sites within the terminator regions of the VA1 gene and with two subunits (TFIIIC220 and TFIIIC110) of human TFIIIC2. Immunodepletion with anti-NF1 antibodies dramatically decreases transcription from the VA1 template in nuclear extract, and mutation at the NF1-binding site in the terminator region of the VA1 gene selectively affects multiple-round transcription (reinitiation of transcription) and termination. In addition, NF1 acts in conjunction with TFIIIC to promote accurate termination by RNA polymerase III on a C-tailed VA1 template.