In the human small nuclear RNA (snRNA) promoters, the presence of a TATA box recognized by the TATA box-binding protein (TBP) determines the selection of RNA polymerase III over RNA polymerase II. The RNA polymerase II snRNA promoters are, therefore, good candidates for TBP-independent promoters. We show here, however, that TBP activates transcription from RNA polymerase II snRNA promoters through a non-TATA box element, the snRNA proximal sequence element (PSE), as part of a new snRNA-activating protein complex (SNAPc). In contrast to the previously identified TBP-containing complexes SL1, TFIID, and TFIIIB, which appear dedicated to transcription by a single RNA polymerase, SNAP c is also essential for RNA polymerase III transcription from the U6 snRNA promoter. The U6 initiation complex appears to contain two forms of TBP, one bound to the TATA box and one bound to the PSE as a part of SNAPc, suggesting that multiple TBP molecules can have different functions within a single promoter. In eukaryotes, transcription is carried out by three different RNA polymerases, none of which can recognize its target promoters directly. Instead, promoter elements are first recognized by specific transcription factors that then recruit the correct RNA polymerase. Because RNA polymerase I, II, and III promoters are generally very different in structure, it has long been assumed that RNA polymerase specificity is achieved through the binding of very distinct sets of transcription factors. Although it is now well established that the TATA box binding protein [TBP) participates in transcription from TATA-containing and TATA-less promoters by all three RNA polymerases, it does so as part of distinct complexes that are each dedicated to transcription by a single RNA polymerase (for review, see Hernandez 1993). Thus, SL1 (Comai et al. 1992), TFIID (for review, see Sawadogo and Sentenac 1990;Roeder 1991; Pugh and Tjian 1992;Zawel and Reinberg 1992,1993), and TFIIIB (Lobo et al. 1992;Simmen et al. 1992b;Taggart et al. 1992; White and Jackson 1992) participate in transcription by RNA polymerases I, II, and III, respectively.The promoters of human small nuclear RNA (snRNA) genes are very similar in structure even though some of them are recognized by RNA polymerase II whereas others are recognized by RNA polymerase III. They therefore serve as a model to study how RNA polymerase specificity is achieved. The human U1 and U2 snRNA promoters are recognized by RNA polymerase II and consist essentially of two elements: A proximal sequence element (PSE) located upstream of position -40, which is essential and sufficient to direct basal levels of transcription, and a distal sequence element (DSE) located upstream of position -200, which serves as a transcriptional enhancer and is characterized by the presence of an octamer motif (for review, see Dahlberg and Lund 1988;Hernandez 1992). The human U6 snRNA promoter, which is recognized by RNA polymerase III, differs from most other RNA polymerase III promoters in that it does not contain any esse...

The TATA-box-binding protein TBP exists in the cell complexed with different sets of TBP-associated factors (TAFs). In general, each of these TBP-TAF complexes is dedicated to transcription by a single RNA polymerase. Thus, SL1, TFIID and TFIIIB are required for transcription by polymerases I, II and III, respectively. Here we characterize a fourth TBP-TAF complex called SNAPc. Unlike the other TBP-TAF complexes, SNAPc is implicated in transcription by two types of polymerases; it is required for transcription of both the RNA polymerase II and III small-nuclear RNA genes and binds specifically to the proximal sequence element PSE, a non-TATA-box basal promoter element common to these two types of genes. In addition to TBP, SNAPc is composed of at least three TAFs, SNAP43, SNAP45 and SNAP50. The predicted amino-acid sequence of SNAP43 reveals that it corresponds to a new protein.

The human homeodomain protein Phox1 interacts functionally with serum response factor (SRF) to impart serum responsive transcriptional activity to SRF-binding sites in a HeLa cell cotransfection assay. However, stable ternary complexes composed of SRF, Phox1, and DNA, which presumably mediate the transcriptional effects of Phox1 in vivo, have not been observed in vitro. Here, we report the identification, purification, and molecular cloning of a human protein that promotes the formation of stable higher-order complexes of SRF and Phox1. We show that this protein, termed SPIN, interacts with SRF and Phox1 in vitro and in vivo. Furthermore, SPIN binds specifically to multiple sequences in the c-fos promoter and interacts cooperatively with Phox1 to promote serum-inducible transcription of a reporter gene driven by the c-fos serum response element (SRE). SPIN is identical to the initiator-binding protein TFII-I. Consistent with this hypothesis, SPINexhibits modest affinity for a characterized initiator sequence in vitro. We propose that this multifunctional protein coordinates the formation of an active promoter complex at the c-fos gene, including the linkage of specific signal responsive activator complexes to the general transcription machinery.

The recruitment of TATA binding protein (TBP) to gene promoters is a critical rate-limiting step in transcriptional regulation for all three eukaryotic RNA polymerases. However, little is known regarding the dynamics of TBP in live mammalian cells. In this report, we examined the distribution and dynamic behavior of green fluorescence protein (GFP)-tagged TBP in live HeLa cells using fluorescence recovery after photobleaching (FRAP) analyses. We observed that GFP-TBP associates with condensed chromosomes throughout mitosis without any FRAP. These results suggest that TBP stably associates with the condensed chromosomes during mitosis. In addition, endogenous TBP and TBP-associated factors (TAFs), specific for RNA polymerase II and III transcription, cofractionated with mitotic chromatin, suggesting that TBP is retained as a TBP-TAF complex on transcriptionally silent chromatin throughout mitosis. In interphase cells, GFP-TBP distributes throughout the nucleoplasm and shows a FRAP that is 100-fold slower than the general transcription factor GFP-TFIIB. This difference supports the idea that TBP and, most likely, TBP-TAF complexes, remain promoter-bound for multiple rounds of transcription. Altogether, our observations demonstrate that there are cell cycle specific characteristics in the dynamic behavior of TBP. We propose a novel model in which the association of TBP-TAF complexes with chromatin during mitosis marks genes for rapid transcriptional activation as cells emerge from mitosis. INTRODUCTIONIn eukaryotic cells, the three RNA polymerases I, II, and III are dedicated to the transcription of distinct classes of genes. Distinct promoter architectures and the assembly of polymerase-specific initiation complexes at gene promoters are keys that dictate the recruitment of the particular class of polymerases. TATA binding protein (TBP) interacts with a variety of TBP-associated factors (TAFs) to form the selectivity factor-1 (SL1), transcription factor TFIID, and TFIIIB complexes that are important for specifying RNA polymerase I, II, and III transcription, respectively (Hernandez, 1993). TBP-TAF complexes are critical players in determining levels of transcription initiation. Thus, the formation of specific TBP-TAF complexes potentially regulates transcription of specific genes under different growth conditions. Increasing the recruitment of these complexes to gene promoters by regulatory proteins is one mechanism for transcriptional activation (Albright and Tjian, 2000;Hampsey and Reinberg, 1999;Hernandez, 1993;Lee and Young, 1998). Once recruited to a promoter, TBP-TAF complexes can perform additional functions that are important for transcriptional regulation, including recruitment of additional members of the general transcriptional machinery to the promoter, induction of conformational changes in DNA topology, and recruitment of coactivator or corepressor proteins that influence gene transcription (reviewed in Tansey and Herr, 1997).The various TBP-TAF complexes have been purified and characterized extensiv...

The human RNA polymerase II and III snRNA promoters have similar enhancers, the distal sequence elements (DSEs), and similar basal promoter elements, the proximal sequence elements (PSEs). The DSE, which contains an octamer motif, binds broadly expressed activator Oct-1. The PSE binds a multiprotein complex referred to as SNAP c or PTF. On DNAs containing both an octamer site and a PSE, Oct-1 and SNAP c bind cooperatively. SNAP c consists of at least four stably associated subunits, SNAP43, SNAP45, SNAP50, and SNAP190. None of the three small subunits, which have all been cloned, can bind to the PSE on their own. Here we report the isolation of cDNAs corresponding to the largest subunit of SNAP c , SNAP190. SNAP190 contains an unusual Myb DNA binding domain consisting of four complete repeats (Ra to Rd) and a half repeat (Rh). A truncated protein consisting of the last two SNAP190 Myb repeats, Rc and Rd, can bind to the PSE, suggesting that the SNAP190 Myb domain contributes to recognition of the PSE by the SNAP complex. SNAP190 is required for snRNA gene transcription by both RNA polymerases II and III and interacts with SNAP45. In addition, SNAP190 interacts with Oct-1. Together, these results suggest that the largest subunit of the SNAP complex is involved in direct recognition of the PSE and is a target for the Oct-1 activator. They also provide an example of a basal transcription factor containing a Myb DNA binding domain.The regulation of transcription initiation is mediated by the interplay between two classes of promoter elements: the basal promoter elements, which can be defined as those promoter elements sufficient to direct basal levels of transcription in vitro, and the regulatory elements, which modulate the levels of transcription. The basal elements are recognized by basal transcription factors, whereas the regulatory elements are recognized by either transcriptional activators or repressors. Eucaryotic activators are often modular, consisting of a DNA binding domain, which targets the activator to the correct promoter, and of activation domains, whose role is to enhance transcription (see references 21-23, 32, and 33 for reviews).The human snRNA gene family contains both RNA polymerase II and RNA polymerase III genes. The RNA polymerase II snRNA promoters consist of a proximal sequence element (PSE), which is sufficient to direct basal levels of transcription in vitro, and a distal sequence element, which activates basal transcription. The RNA polymerase III snRNA promoters are similar, except that basal transcription is directed by the combination of a PSE and a TATA box (reviewed in reference 9). The PSE is recognized by a multisubunit complex called the SNAP complex (SNAP c ) (7) or PTF (34). Since SNAP c can bind to the PSE on its own, it corresponds to a sequence-specific DNA binding basal transcription factor. SNAP c contains at least four subunits, SNAP43, SNAP45, SNAP50, and SNAP190, and cDNAs encoding the SNAP43 (7) or PTF ␥ (35), SNAP45 (24) or PTF ␦ (35), and SNAP50 (6) or PTF ␤ (2) su...