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...

In eukaryotes, activation of transcription involves an interplay between activators bound to cis-regulatory elements and factors bound to basal elements near the start site of transcription. The basal elements, for example the TATA box or proximal sequence element (PSE) of small nuclear RNA (snRNA) promoters, nucleate the assembly of basal transcription complexes, components of which interact with activators. Although one basal transcription complex can interact with many activators, it is unclear whether different basal transcription complexes can direct different responses to particular activators. We show here that changing the arrangement of basal elements can alter the response to transcriptional activation domains. Indeed, in the human U6 snRNA promoter, point mutation of either a TATA box or PSE results in diametrically opposed responses to VP16- and Sp1-derived activation domains. These basal elements can even discriminate small changes in an activation domain. Thus the arrangement of basal promoter elements provides a mechanism for differential regulation of transcription.

The octamer motif is a common cis-acting regulatory element that functions in the transcriptional control regions of diverse genes and in viral origins of replication. The , were present at low levels in oocytes and early embryos and were dramatically upregulated during early gastrulation. In contrast to the Oct-60 mRNA, translation of Oct-25 mRNA appeared to be developmentally regulated, since the corresponding protein was detected in embryos during gastrulation but not in oocytes or rapidly cleaving embryos. Transcripts from the third POU protein gene, Oct-91, were induced after the midblastula transition and reached their highest levels of accumulation during late gastrulation. The expression of all three genes decreased during late gastrulation and early neurulation. By analogy with other members of the POU-domain gene family, the products of these genes may play critical roles in the determination of cell fate and the regulation of cell proliferation.

Human U6 small nuclear RNA (snRNA) gene transcription by RNA polymerase III requires cooperative promoter binding involving the snRNA-activating protein complex (SNAP c ) and the TATA-box binding protein (TBP). To investigate the role of SNAP c for TBP function at U6 promoters, TBP recruitment assays were performed using full-length TBP and a mini-SNAP c containing SNAP43, SNAP50, and a truncated SNAP190. Mini-SNAP c efficiently recruits TBP to the U6 TATA box, and two SNAP c subunits, SNAP43 and SNAP190, directly interact with the TBP DNA binding domain. Truncated SNAP190 containing only the Myb DNA binding domain is sufficient for TBP recruitment to the TATA box. Therefore, the SNAP190 Myb domain functions both to specifically recognize the proximal sequence element present in the core promoters of human snRNA genes and to stimulate TBP recognition of the neighboring TATA box present in human U6 snRNA promoters. The SNAP190 Myb domain also stimulates complex assembly with TBP and Brf2, a subunit of a snRNA-specific TFIIIB complex. Thus, interactions between the DNA binding domains of SNAP190 and TBP at juxtaposed promoter elements define the assembly of a RNA polymerase IIIspecific preinitiation complex.Transcription in eukaryotic organisms occurs by three different RNA polymerases that transcribe different classes of genes. The recruitment of a specific RNA polymerase to a given promoter is dictated by the nature of the preinitiation complex (1-4); however, the molecular determinants for RNA polymerase specificity are not known. One possibility is that recruitment of TBP 1 complexes containing distinct cadres of TBPassociated factors (TAFs) may confer polymerase specificity. For example, the TBP complexes SL1, TFIID, and TFIIIB are multiprotein TBP⅐TAF complexes that are required for transcription by RNA polymerases I, II, and III, respectively (for review, see Refs. 5-7). These various TBP⅐TAF complexes play crucial roles by serving as targets for regulatory proteins and providing specific promoter recognition functions. Once recruited to a promoter, TBP⅐TAF complexes further provide an important structural façade during preinitiation complex assembly to recruit other general transcription factors dedicated to transcription by a specific RNA polymerase.In contrast to the well characterized TBP complexes that function for transcription of most genes, how TBP is recruited to human small nuclear (sn) RNA gene promoters is understood less well. None of the aforementioned TBP⅐TAF complexes appears to function at these genes (8, 9). Human snRNA genes are unusual because they have similar promoters, and yet some snRNA genes are transcribed by RNA polymerase II (e.g. U1) and others by RNA polymerase III (e.g. U6) (for review, see Refs. 10 and 11). Thus, these genes serve as an important model for understanding the mechanisms of polymerase specificity (12). Each gene contains a proximal sequence element (PSE) in the core promoter which recruits the general transcription factor SNAP c (9), which is also known as proxi...

Extraction of DNA from plant tissue is often problematic, as many plants contain high levels of secondary metabolites that can interfere with downstream applications, such as the PCR. Removal of these secondary metabolites usually requires further purification of the DNA using organic solvents or other toxic substances. In this study, we have compared two methods of DNA purification: the cetyltrimethylammonium bromide (CTAB) method that uses the ionic detergent hexadecyltrimethylammonium bromide and chloroform-isoamyl alcohol and the Edwards method that uses the anionic detergent SDS and isopropyl alcohol. Our results show that the Edwards method works better than the CTAB method for extracting DNA from tissues of Petunia hybrida. For six of the eight tissues, the Edwards method yielded more DNA than the CTAB method. In four of the tissues, this difference was statistically significant, and the Edwards method yielded 27-80% more DNA than the CTAB method. Among the different tissues tested, we found that buds, 4 days before anthesis, had the highest DNA concentrations and that buds and reproductive tissue, in general, yielded higher DNA concentrations than other tissues. In addition, DNA extracted using the Edwards method was more consistently PCR-amplified than that of CTAB-extracted DNA. Based on these results, we recommend using the Edwards method to extract DNA from plant tissues and to use buds and reproductive structures for highest DNA yields.