Proximal Sequence Element-Binding Transcription Factor (PTF) Is a Multisubunit Complex Required for Transcription of both RNA Polymerase II- and RNA Polymerase III-Dependent Small Nuclear RNA Genes

Abstract

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...

Identification of an autonomously initiating RNA polymerase III holoenzyme containing a novel factor that is selectively inactivated during protein synthesis inhibition

“…However, whereas purified TDF completely restored transcription of the subclass 1 (5S RNA) and subclass 2 (tRNA and VA RNA) genes, it only partially restored transcription of subclass 3 (U6 and 7SK RNA) genes. The latter result suggests that additional subclass 3-specific factors also are partially inactivated by cycloheximide treatment and are consistent with previous demonstrations (for review, see Yoon et al 1995) of such factors. Nevertheless, these results indicate that TDF is a general transcription factor essential for transcription of all three subclasses of class III genes.…”

Different Functional Modes of p300 in Activation of RNA Polymerase III Transcription from Chromatin Templates

“…Importantly, ChIP analyses showed that, in cells, p300 is targeted to actively transcribed tRNA genes, as well as to U6 RNA genes that do not contain binding sites for TFIIIC. It is possible, therefore, that promoter recognition factor SNAPc/PTF (42,54,73) or Oct-1 (55,59) is involved in recruiting p300 to the U6 gene.…”

Characterization of a Drosophila Proximal‐Sequence‐Element‐Binding Protein Involved in Transcription of Small Nuclear RNA Genes

“…At least four polypeptides with reported molecular masses of approximately 180-200, 50-55, 44-45 and 43-45 kDa have been identified as common components of SNAPc and PTF (Henry et al, 1995;Yoon et al, 1995). The TATA-box-binding protein (TBP), which is required for snRNA transcription by both RNAP 11 and RNAP I11 (Margottin et al, 1991 ; Lob0 et al., 1991;Simmen et al, 1991;Zamrod et al, 1993;Heard et al, 1993; Bernds et al, 1993;Sadowski et al, 1993), was considered to be an integral subunit of SNAPc (Sadowski et al, 1993;Henry et al, 1995), but was not detected at significant levels in purified PTF (Yoon et al, 1995;Yoon and Roeder, 1996). Except for this difference, PTF and SNAPc appear to be identical.…”

“…Different research groups have termed it the PSE-binding protein (PBP), proximal transcription factor (PTF), or small nuclear RNA activator protein complex (SNAPc) (Waldschmidt et al, 1991;Murphy et al, 1992;Simmen et al, 1992;Sadowski et al, 1993). It is capable of activating U1 and U2 gene transcription by RNAP 11, as well as U6 and 7SK gene transcription by RNAP I11 (Sadowski et al, 1993;Henry et al, 1995;Yoon et al, 1995). At least four polypeptides with reported molecular masses of approximately 180-200, 50-55, 44-45 and 43-45 kDa have been identified as common components of SNAPc and PTF (Henry et al, 1995;Yoon et al, 1995).…”