Abstract: The TATA box binding protein (TBP) has been called the universal transcription factor. In eukaryotes, three different multisubunit nuclear RNA polymerases transcribe three distinct classes of RNAs. RNA polymerase II (Pol II) transcribes protein-coding genes. RNA polymerases I and III (Pol I and Pol III) transcribe ribosomal RNAs and small stable RNAs such as tRNAs, respectively. Each RNA polymerase requires distinct additional general transcription factors to locate promoters and initiate transcription. TBP is the one general transcription factor required by all three RNA polymerases (1Burley S.K Roeder R.G Annu. Rev. Biochem. 1996; 65: 769-799Crossref PubMed Scopus (610) Google Scholar). For Pol II, TBP binds directly to the TATA box promoter element, where it nucleates assembly of Pol II and general transcription factors TFIIA, B, D, E, F, and H into a preinitiation complex. This TBP–TATA box interaction defines the transcription start site and therefore the precise DNA sequence translated into protein. Furthermore, the affinity of TBP for a gene's specific TATA box sequence contributes to promoter strength, i.e., the frequency with which the gene is transcribed. Interestingly, recent genomic and cDNA sequencing has revealed that all multicellular animals from nematodes to humans actually express two TBP-like proteins, TBP itself and a second protein called TLF, for T BP-l ike f actor. Initially called TLP (11Ohbayashi T Makino Y Tamura T Nucleic Acids Res. 1999; 27: 750-755Crossref PubMed Scopus (75) Google Scholar), TRF2 (14Rabenstein M.D Zhou S Lis J.T Tjian R Proc. Natl. Acad. Sci. USA. 1999; 96: 4791-4796Crossref PubMed Scopus (140) Google Scholar, 17Teichmann M Wang Z Martinez E Tjernberg A Zhang D Vollmer F Chait B.T Roeder R.G Proc. Natl. Acad. Sci. USA. 1999; 96: 13720-13725Crossref PubMed Scopus (99) Google Scholar), TRF (9Maldonado E J. Biol. Chem. 1999; 274: 12963-12966Crossref PubMed Scopus (31) Google Scholar), or TRP (10Moore P.A Ozer J Salunek M Jan G Zerby D Campbell S Lieberman P.M Mol. Cell. Biol. 1999; 19: 7610-7620Crossref PubMed Scopus (79) Google Scholar), sequence comparisons suggest that the TLFs are all orthologs of each other with a function distinct from TBPs (3Dantonel J.-C Wurtz J.-M Poch O Moras D Tora L Trends Biochem. Sci. 1999; 285: 335-339Abstract Full Text Full Text PDF Scopus (86) Google Scholar). Drosophila is a notable exception in that it expresses a third TBP-related factor, TRF1 (2Crowley T.E Hoey T Liu J.K Jan Y.N Jan L.Y Tjian R Nature. 1993; 361: 557-561Crossref PubMed Scopus (101) Google Scholar). Sequence analysis indicates that Drosophila TRF1 is closely related to the TBPs of other animals, including the more abundant Drosophila TBP initially studied. Recent papers have now shed light on the functions of these TBP-related and TBP-like factors. 16Takada S Lis J.T Zhou S Tjian R Cell. 2000; 101: 459-469Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar has revealed that Drosophila TRF1 substitutes for TBP in RNA polymerase III transcription. And two studies appearing in the September issue of Molecular Cell (4Dantonel J.-C Quintin S Lakatos L Labouesse M Tora L Mol. Cell. 2000; 6: 715-722Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 8Kaltenbach L Horner M.A Rothman J.H Mango S.E Mol. Cell. 2000; 6: 705-713Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) have used RNA interference to analyze the function of C. elegans TLF in early development, revealing that TLF is generally required for cellular differentiation. The ∼180 amino acid saddle-shaped DNA binding core of TBPs from multiple organisms, including Drosophila TRF1, are closely related, each containing identical amino acids at positions critical to the domain's structure (1Burley S.K Roeder R.G Annu. Rev. Biochem. 1996; 65: 769-799Crossref PubMed Scopus (610) Google Scholar) and DNA binding specificity (12Patikoglou G.A Kim J.L Sun L Yang S.H Kodadek T Burley S.K Genes Dev. 1999; 13: 3217-3230Crossref PubMed Scopus (226) Google Scholar) (Figure 1, red and black highlights). Similarly, TLFs have ∼180 amino acid domains that are about 60% similar to the core domain of TBPs, with identical or chemically similar amino acids at many equivalent positions (Figure 1, black and gray highlights). This sequence similarity suggests that a TLF core domain folds into a saddle-shaped structure similar to the TBP core. But, as pointed out by 3Dantonel J.-C Wurtz J.-M Poch O Moras D Tora L Trends Biochem. Sci. 1999; 285: 335-339Abstract Full Text Full Text PDF Scopus (86) Google Scholar, the TLFs contain distinct, conserved amino acids at several positions that distinguish them from TBPs (Figure 1, yellow highlight). The conserved differences between TLFs and TBPs include amino acids that would likely alter the DNA binding specificity of TLFs compared to TBPs (Figure 2; 3Dantonel J.-C Wurtz J.-M Poch O Moras D Tora L Trends Biochem. Sci. 1999; 285: 335-339Abstract Full Text Full Text PDF Scopus (86) Google Scholar, 12Patikoglou G.A Kim J.L Sun L Yang S.H Kodadek T Burley S.K Genes Dev. 1999; 13: 3217-3230Crossref PubMed Scopus (226) Google Scholar). Importantly, pairs of phenylalanines that intercalate between base pairs 1 and 2, and 7 and 8 of the TATA box, bending the DNA sharply at these positions in the TBP–TATA box complex, are not conserved in TLFs (Figure 1, arrowheads). Consistent with this, most reports indicate that recombinant human TLF fails to bind to TATA boxes (10Moore P.A Ozer J Salunek M Jan G Zerby D Campbell S Lieberman P.M Mol. Cell. Biol. 1999; 19: 7610-7620Crossref PubMed Scopus (79) Google Scholar, 14Rabenstein M.D Zhou S Lis J.T Tjian R Proc. Natl. Acad. Sci. USA. 1999; 96: 4791-4796Crossref PubMed Scopus (140) Google Scholar, 17Teichmann M Wang Z Martinez E Tjernberg A Zhang D Vollmer F Chait B.T Roeder R.G Proc. Natl. Acad. Sci. USA. 1999; 96: 13720-13725Crossref PubMed Scopus (99) Google Scholar). On the other hand, very few of the conserved differences between TLFs and TBPs occur in amino acids of TBP involved in protein–protein interactions with TFIIA, TFIIB, or Pol II or Pol III TBP-associated factors (TAFs) (Figure 2; Bryant et al., 1996 and references therein; 15Shen Y Kassavetis G.A Bryant G.O Berk A.J Mol. Cell. Biol. 1998; 18: 1692-1700Crossref PubMed Scopus (42) Google Scholar, 3Dantonel J.-C Wurtz J.-M Poch O Moras D Tora L Trends Biochem. Sci. 1999; 285: 335-339Abstract Full Text Full Text PDF Scopus (86) Google Scholar). Accordingly, human and Drosophila TLF bind TFIIA and TFIIB in vitro (10Moore P.A Ozer J Salunek M Jan G Zerby D Campbell S Lieberman P.M Mol. Cell. Biol. 1999; 19: 7610-7620Crossref PubMed Scopus (79) Google Scholar, 14Rabenstein M.D Zhou S Lis J.T Tjian R Proc. Natl. Acad. Sci. USA. 1999; 96: 4791-4796Crossref PubMed Scopus (140) Google Scholar, 17Teichmann M Wang Z Martinez E Tjernberg A Zhang D Vollmer F Chait B.T Roeder R.G Proc. Natl. Acad. Sci. USA. 1999; 96: 13720-13725Crossref PubMed Scopus (99) Google Scholar). These similarities and differences in the structures of TBPs and TLFs have led to the suggestion that TLF might nucleate the assembly of Pol II preinitiation complexes on a class of genes with a promoter element distinct from a TATA box. Initial studies of Drosophila TRF1 suggested that it might function in the transcription of a restricted set of tissue-specific genes by Pol II (6Hansen S.K Takada S Jacobson R.H Lis J.T Tjian R Cell. 1997; 91: 71-83Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). However, as reported in this initial study, in situ localization of TRF1 on salivary gland polytene chromosomes showed that TRF1 was associated with many loci containing tRNA genes. This led 16Takada S Lis J.T Zhou S Tjian R Cell. 2000; 101: 459-469Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar to analyze the function of TRF1 in Pol III transcription. This study presents compelling evidence that Drosophila TRF1 replaces TBP in the Drosophila version of the Pol III transcription factor TFIIIB. In yeast, TFIIIB, which is required for transcription of all genes transcribed by RNA polymerase III, is composed of TBP and two pol III–specific TAFs, one of which is called BRF for TFII B-r elated f actor (White, 1988). Immunodepletion of TRF1 from Drosophila nuclear extract virtually eliminated in vitro transcription of several classes of RNA polymerase III transcribed genes tested, whereas immunodepletion of TBP had little effect. A Drosophila homolog of the BRF TFIIIB subunit characterized in yeast and humans (18White R.J RNA Polymerase III Transcription. Second Edition. Springer-Verlag, Berlin1998Crossref Google Scholar) was found to coprecipitate with TRF1, and its cDNA was cloned. Importantly, immunoprecipitation of Drosophila nuclear extract with antibodies to TRF1 and BRF demonstrated that >90% of TRF1 is associated with BRF. TRF1 associated with BRF in vitro, whereas Drosophila TBP did not. And a TRF1-Drosophila BRF1 complex prepared from the purified recombinant proteins reconstituted transcription of RNA polymerase III-transcribed genes in extracts immunodepleted with antibody to either TRF1 or BRF. Finally, BRF and TRF1 colocalized on salivary gland polytene chromosomes at sites that correspond to clusters of tRNA and tandemly repeated 5S rRNA genes. These results indicate that in Drosophila (and probably other insects), a specialized form of TBP, TRF1, evolved from a duplicated TBP gene to perform the functions of TBP in Pol III transcription. Analysis of the virtually complete Drosophila genome sequence has not revealed any additional TBP-related factors. Therefore, it seems likely that Pol I transcription in Drosphila will involve either TBP or the closely related TRF1, but at this point, it is not clear which. In addition to its function in Pol III transcription, there are hints that Drosophila TRF1 might also participate in the transcription of a specialized class of Pol II genes, as originally proposed (2Crowley T.E Hoey T Liu J.K Jan Y.N Jan L.Y Tjian R Nature. 1993; 361: 557-561Crossref PubMed Scopus (101) Google Scholar, 6Hansen S.K Takada S Jacobson R.H Lis J.T Tjian R Cell. 1997; 91: 71-83Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). In the salivary gland polytene chromosome in situ staining studies reported by 16Takada S Lis J.T Zhou S Tjian R Cell. 2000; 101: 459-469Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, multiple loci were stained by anti-TRF1 but not anti-BRF antibody. Since BRF is required for Pol III transcription, this result suggests that TRF1 is performing a different function at these loci. In vitro transcription and in vivo transfection assays with the tudor promoter also are consistent with this possibility (7Holmes, M.C., and Tjian, R. (2000). Promoter-selective properties of the TBP-related factor TRF1. Science 288, 867–870.Google Scholar). However, it is possible that TRF1 was detected with greater sensitivity than BRF in these studies, and that BRF was in fact present at sites where TRF1 alone was detected. TRF1, which is related very closely to other TBPs (Figure 1), can bind to a TATA box in the tudor promoter, interact with TFIIB, and direct transcription by Pol II in vitro (6Hansen S.K Takada S Jacobson R.H Lis J.T Tjian R Cell. 1997; 91: 71-83Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Consequently, it might be expected to do so in vivo when expressed at high levels by transient transfection. As yet, the question remains open as to whether TRF1 normally is involved in transcription by Pol II in vivo. The first clues concerning the biological function of TLFs are presented in the current Molecular Cell papers by 4Dantonel J.-C Quintin S Lakatos L Labouesse M Tora L Mol. Cell. 2000; 6: 715-722Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar and 8Kaltenbach L Horner M.A Rothman J.H Mango S.E Mol. Cell. 2000; 6: 705-713Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar. Their studies in C. elegans use RNA interference (RNAi) to eliminate expression of a specific gene. In RNAi, double-stranded RNA prepared from a specific gene is injected into adult hermaphrodites, resulting in degradation of the equivalent RNA in progeny worms (5Fire A Trends Genet. 1999; 15: 358-363Abstract Full Text Full Text PDF PubMed Scopus (480) Google Scholar). Both groups observed the remarkable result that most embryos from mothers injected with C. elegans double-stranded TLF-RNAi (tlf(RNAi) embryos) arrest as clusters of 80–350 undifferentiated cells that fail to undergo even the earliest step in gastrulation. Analysis of gene expression in most of these terminal phenotype embryos revealed a general failure of expression of differentiation markers for virtually all the cell lineages of a normal embryo. 4Dantonel J.-C Quintin S Lakatos L Labouesse M Tora L Mol. Cell. 2000; 6: 715-722Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar also observed a reduction in the level of phosphorylated RNA polymerase II, consistent with a generalized decrease in Pol II transcription in these terminal phenotype embryos. 4Dantonel J.-C Quintin S Lakatos L Labouesse M Tora L Mol. Cell. 2000; 6: 715-722Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar also noted a rare class of tlf(RNAi) embryos with a terminal phenotype of less than 80 cells that expressed several genes not normally expressed in 80-cell embryos. Since a dramatic decrease in the level of TLF led to the expression of multiple genes not normally expressed, Dantonel et al. suggested that TLF might act as a general repressor of transcription in the early embryo. This hypothesis is consistent with the observation that human TLF inhibits in vitro transcription in reactions with TBP or TFIID, perhaps by competing for other general transcription factors such as TFIIA and TFIIB (10Moore P.A Ozer J Salunek M Jan G Zerby D Campbell S Lieberman P.M Mol. Cell. Biol. 1999; 19: 7610-7620Crossref PubMed Scopus (79) Google Scholar, 17Teichmann M Wang Z Martinez E Tjernberg A Zhang D Vollmer F Chait B.T Roeder R.G Proc. Natl. Acad. Sci. USA. 1999; 96: 13720-13725Crossref PubMed Scopus (99) Google Scholar). Alternatively, it could be that partial inhibition of TLF in this rare class of embryos interfered with cell division, but not with developmental programs leading to transcription of the genes assayed. To determine if the observed failure to express differentiation-specific genes in most of the terminal phentoype embryos was due to a general defect in early embryonic transcription, 8Kaltenbach L Horner M.A Rothman J.H Mango S.E Mol. Cell. 2000; 6: 705-713Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar analyzed tlf(RNAi) embryos before the terminal phenotype occurred. Of three genes analyzed that are normally expressed at gastrulation, the expression of only one, end-1, was severely inhibited. Transcripton of another gene, pes-10, a gene of unknown function that is among the earliest genes transcribed in the embryo, was also defective in tlf(RNAi) embryos at an even earlier time in embryogenesis. The observation that transcription of some genes (end-1 and pes-10) is severely defective in tlf(RNAi) embryos, but that of other genes normally transcribed at gastrulation is not, led Kaltenbach et al. to conclude that TLF is required for the transcription of a subset of genes in the early embryo. The observation that Drosophila TLF is associated with a much smaller number of loci on salivary gland polytene chromosomes than TFIID TAF250 (14Rabenstein M.D Zhou S Lis J.T Tjian R Proc. Natl. Acad. Sci. USA. 1999; 96: 4791-4796Crossref PubMed Scopus (140) Google Scholar) is also consistent with the model that TLF is required for the transcription of a specific subset of genes in differentiated cells as well. The failure to express end-1 may explain the defect in gastrulation in tlf(RNAi) embryos. This gene encodes a GATA transcription factor expressed uniquely in the blastomeres that develop into endoderm, including the cells whose movements initiate gastrulation. As Kaltenbach et al. discuss, there is evidence that expression of either end-1 or a closely related gene, end-3, expressed at the same time as end-1 in early embryogenesis, is required for the differentiation of endodermal precursor cells and the initiation of gastrulation. If tlf(RNAi) embryos failed to express end-3 as well as end-1, they would not be expected to undergo the cell movements of gastrulation. Kaltenbach et al. also analyzed transcription in four-cell embryos, when embryonic transcription is first detected in C. elegans by performing in situ immunostaining experiments using antibodies that recognize phosphorylated forms of Pol II. The largest subunit of Pol II is reversibly phosphorylated at multiple sites in its C-terminal domain (CTD) where there are several repeats of a seven amino acid sequence. Pol II is assembled into a preinitiation complex at a promoter with an unphosphorylated CTD. The CTD then becomes phosphorylated at multiple sites during transcription initiation and the initial period of transcription. Kaltenbach et al. stained embryos with monoclonal antibody H5 that binds to the CTD phosphorylated at serine 2 of the repeat sequence (13Patturajan M Schulte R.J Sefton B.M Berezney R Vincent M Bensaude O Warren S.L Corden J.L J. Biol. Chem. 1998; 273: 4689-4694Crossref PubMed Scopus (212) Google Scholar). H5 reactivity was delayed and weak in tlf(RNAi) embryos, leading the authors to conclude that TLF is required for general Pol II transcription at this very early stage of embryogenesis. However, currently the relation between H5 immunoreactivity and general Pol II transcription is not clear. Phosphorylation of the CTD by the general transcription factor TFIIH during Pol II initiation occurs at serine 5 in the repeated sequence, not the serine 2 position detected by monoclonal antibody H5. It is not yet clear what kinase is principally responsible for the position 2 phosphorylation. The only kinase known to do so is the Cdk9-CyclinT heterodimer of the transcription elongation factor pTEFb which stimulates transcription elongation in vitro (19Zhou M Halanski M.A Radonovich M.F Kashanchi F Peng J Price D.H Brady J.N Mol. Cell. Biol. 2000; 20: 5077-5086Crossref PubMed Scopus (215) Google Scholar). The observation that tlf(RNAi) embryos at the four- to eight-cell stage have reduced phosphorylation of the CTD at serine 2 in all nuclei compared to wild-type is very interesting. But it seems premature to conclude that this indicates a general defect in all Pol II transcription at this stage of embryogenesis. 4Dantonel J.-C Quintin S Lakatos L Labouesse M Tora L Mol. Cell. 2000; 6: 715-722Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar also observed decreased reactivity to H5 in terminal phenotype embryos, and further, that the large Pol II subunit had the high mobility in an SDS gel indicative of hypophosphorylation throughout the length of the CTD. But it was not clear whether this could have been simply a consequence of the death of the terminal phenotype embryos. As Kaltenbach et al. discuss, the failure to express a subset of specific genes at the time of gastrulation may indicate that TLF is directly required for their transcription, or that TLF is indirectly required because it's necessary for the expression of another required gene, such as a specific transcription factor. Using in situ methods, they present evidence that TLF binds directly to a 300 bp pes-10 promoter region fragment. This result is consistent with the model that TLF functions directly to control pes-10 transcription. The papers by 4Dantonel J.-C Quintin S Lakatos L Labouesse M Tora L Mol. Cell. 2000; 6: 715-722Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar and 8Kaltenbach L Horner M.A Rothman J.H Mango S.E Mol. Cell. 2000; 6: 705-713Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar demonstrate that TLF is an essential transcription factor required for transcription of a restricted set of specific genes in the early C. elegans embryo. One or more of these TLF-dependent genes is required for the viability of embryonic cells and the differentiation of virtually all cell lineages. Now that it is clear that TLF is essential for cellular differentiation, a number of significant questions arise. What genes transcribed during embryogenesis besides pes-10 are direct targets of TLF? How do they contribute to differentiation? Are end-1 and other key transcription factors that act as developmental regulators for all the cell lineages in the early embryo direct TLF targets? Since TLF is expressed ubiquitously throughout the life cycles of metazoans, what genes does it control in differentiated cells? The answer to this question will likely require the isolation of conditional mutants of TLF, such as temperature-sensitive mutants, to allow the inactivation of TLF once early stages in embryogenesis are complete. Another interesting question concerns how TLF functions in transcription. Since TLF is closely related to TBP and can bind TFIIA and TFIIB in vitro (10Moore P.A Ozer J Salunek M Jan G Zerby D Campbell S Lieberman P.M Mol. Cell. Biol. 1999; 19: 7610-7620Crossref PubMed Scopus (79) Google Scholar, 14Rabenstein M.D Zhou S Lis J.T Tjian R Proc. Natl. Acad. Sci. USA. 1999; 96: 4791-4796Crossref PubMed Scopus (140) Google Scholar, 17Teichmann M Wang Z Martinez E Tjernberg A Zhang D Vollmer F Chait B.T Roeder R.G Proc. Natl. Acad. Sci. USA. 1999; 96: 13720-13725Crossref PubMed Scopus (99) Google Scholar) an obvious model is that TLF replaces TBP in the assembly of general transcription factors and RNA polymerase II into a transcription preinitiation complex on TLF-dependent promoters. Since TLF does not bind to TATA boxes, the simplest version of this model predicts that TLF-dependent promoters would have a TLF binding site in place of a TATA box near the transcription start site. However, the one promoter to which TLF has been shown to bind in vivo, the pes-10 promoter, does have a potential TATA box close to the transcription start site, although end-1, another TLF-dependent gene, does not (8Kaltenbach L Horner M.A Rothman J.H Mango S.E Mol. Cell. 2000; 6: 705-713Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). At present, there is no data to indicate whether TLF functions in the absence of TBP or in conjunction with TBP. It is even possible that TLF functions similarly to other regulatory transcription factors with distinct DNA binding and activation domains, but with a DNA binding domain similar to the TBP core and assisted by TFIIA and TFIIB. However, this seems unlikely given the close similarity of TLFs and TBPs. In addressing this question, it would be interesting to determine the TLF binding site(s) in the pes-10 300 bp promoter fragment studied by Kaltenbach et al. and in other direct targets of TLF. Are they within a few tens of base pairs of the transcription start sites? If insertions or deletions of several base pairs were made between a TLF binding site and the transcription start site, would the start site shift accordingly, as in the classic experiments that established that TATA boxes determine transcription start sites? It is remarkable that TLFs have been found in multicellular animals, but not in fungi or plants. It has been suggested that TBP-like factors might function like sigma factors in bacteria that direct RNA polymerase to specific classes of genes. However, so far, genomic sequencing has revealed only two TBP-like factors in most metazoans (except Drosophila), rather than the five to ten sigma factors in a single species of bacteria. If TBP-like factors function as sigma factors do in bacteria, it would seem that there should be more TBP-like factors in multicellular animals, and that they would be found in fungi and plants as well as metazoans. Rather, it seems that TLF evolved early in metazoans, probably from a duplication of a TBP gene, to perform some function that cannot be accomplished by TBP and is required for cellular differentiation. It will be of considerable interest to learn more precisely what that function is.