Transcriptional mechanisms remain poorly understood in trypanosomatid protozoa. In particular, there is no knowledge about the function of basal transcription factors, and there is an apparent rarity of promoters for protein-coding genes transcribed by RNA polymerase (Pol) II. Here we describe a Trypanosoma brucei factor related to the TATA-binding protein (TBP). Although this TBP-related factor (TBP-related factor 4 [TRF4]) has about 31% identity to the TBP core domain, several key residues involved in TATA box binding are not conserved. Depletion of the T. brucei TRF4 (TbTRF4) by RNA interference revealed an essential role in RNA Pol I, II, and III transcription. Using chromatin immunoprecipitation, we further showed that TRF4 is recruited to the Pol I-transcribed procyclic acidic repetitive genes, Pol II-transcribed spliced leader RNA genes, and Pol III-transcribed U-snRNA and 7SL RNA genes, thus supporting a role for TbTRF4 in transcription performed by all three nuclear RNA polymerases. Finally, a search for TRF4 binding sites in the T. brucei genome led to the identification of such sites in the 3′ portion of certain protein-coding genes, indicating a unique aspect of Pol II transcription in these organisms.
TATA binding protein (TBP) is a key component of the eukaryotic transcription initiation machinery. It functions in several complexes involved in core promoter recognition and assembly of the pre-initiation complex. Through gene duplication eukaryotes have expanded their repertoire of TATA binding proteins, leading to a variable composition of the transcription machinery. In vertebrates this repertoire consists of TBP, TBP-like factor (TLF, also known as TBPL1, TRF2) and TBP2 (also known as TBPL2, TRF3). All three factors are essential, with TLF and TBP2 playing important roles in development and differentiation, in particular gametogenesis and early embryonic development, whereas TBP dominates somatic cell transcription. TBP-related factors may compete for promoters when co-expressed, but also show preferential interactions with subsets of promoters. Initiation factor switching occurs on account of differential expression of these proteins in gametes, embryos and somatic cells. Paralogs of TFIIA and TAF subunits account for additional variation in the transcription initiation complex. This variation in core promoter recognition accommodates the expanded regulatory capacity and specificity required for germ cells and embryonic development in higher eukaryotes.
Leishmania parasites (order Kinetoplastida, family Trypanosomatidae) cause a spectrum of human diseases ranging from asymptomatic to lethal. The ∼33.6 Mb genome is distributed among 36 chromosome pairs that range in size from ∼0.3 to 2.8 Mb. The complete nucleotide sequence of Leishmania major Friedlin chromosome 1 revealed 79 protein-coding genes organized into two divergent polycistronic gene clusters with the mRNAs transcribed towards the telomeres. We report here the complete nucleotide sequence of chromosome 3 (384 518 bp) and an analysis revealing 95 putative protein-coding ORFs. The ORFs are primarily organized into two large convergent polycistronic gene clusters (i.e. transcribed from the telomeres). In addition, a single gene at the left end is transcribed divergently towards the telomere, and a tRNA gene separates the two convergent gene clusters. Numerous genes have been identified, including those for metabolic enzymes, kinases, transporters, ribosomal proteins, spliceosome components, helicases, an RNA-binding protein and a DNA primase subunit.
The core human U6 promoter consists of a proximal sequence element (PSE) located upstream of a TATA box. The PSE is recognized by the snRNA-activating protein complex (SNAPc), which consists of five types of subunits, SNAP190, SNAP50, SNAP45, SNAP43, and SNAP19. The TATA box is recognized by TATA box binding protein (TBP). In addition, basal U6 transcription requires the SANT domain protein Bdp1 and the transcription factor IIB-related factor Brf2. SNAPc and mini-SNAPc, which consists of just SNAP43, SNAP50, and the N-terminal third of SNAP190, bind cooperatively with TBP to the core U6 promoter. By generating complexes smaller than mini-SNAPc, we have identified a 50-amino-acid region within SNAP190 that is (i) required for cooperative binding with TBP in the context of mini-SNAPc and (ii) sufficient for cooperative binding with TBP when fused to a heterologous DNA binding domain. We show that derivatives of mini-SNAPc lacking this region are active for transcription and that with such complexes, TBP can still be recruited to the U6 promoter through cooperative interactions with Brf2. Our results identify complexes smaller than mini-SNAPc that are transcriptionally active and show that there are at least two redundant mechanisms to stably recruit TBP to the U6 transcription initiation complex.
TATA-box-binding protein (TBP)-related factor 3, TRF3 (also called TBP2), is a vertebrate-specific member of the TBP family that has a conserved C-terminal region and DNA binding domain virtually identical to that of TBP1. TRF3 is highly expressed during embryonic development, and studies in zebrafish and Xenopus have shown that TRF3 is required for normal embryogenesis2,3. Here we show that Trf3-depleted zebrafish embryos exhibit multiple developmental defects and, in particular, fail to undergo hematopoiesis. Expression profiling for Trf3-dependent genes identified mespa, which encodes a transcription factor whose murine orthologue is required for mesoderm specification4, and chromatin immunoprecipitation verified that Trf3 binds to the mespa promoter. Depletion of Mespa resulted in developmental and hematopoietic defects strikingly similar to those induced by Trf3 depletion. Injection of mespa mRNA restored normal development to a Trf3-depleted embryo, indicating mespa is the single Trf3 target gene required for zebrafish embryogenesis. Zebrafish embryos depleted of Trf3 or Mespa also failed to express cdx4, a caudal-related gene required for hematopoiesis. Mespa binds to the cdx4 promoter, and epistasis analysis revealed an ordered trf3-mespa-cdx4 pathway. Thus, in zebrafish commitment of mesoderm to the hematopoietic lineage occurs through a transcription factor pathway initiated by a TBP-related factor.
Transcription of the TATA box binding protein (TBP) gene in Acanthamoeba castellanii is regulated by TATA box binding protein promoter binding factor (TPBF), which binds to an upstream TBP promoter element to stimulate transcription, and to a TATA proximal element, where it represses transcription. In order to extend these observations to the in vivo chromatin context, the TBP gene was examined by in situ footprinting and chromatin immunoprecipitation (ChIP). Acanthamoeba DNA is nucleosomal with a repeat of ∼160 bp, and an intranucleosomal DNA periodicity of 10.5 bp. The TBP gene comprises a 220 bp micrococcal nuclease hypersensitive site corresponding to the promoter regulatory elements previously identified, flanked by protected regions of a size consistent with the presence of nucleosomes. ChIP data indicated that TPBF is associated with the TBP, TPBF and MIL gene promoters, but not to the CSP21, MIIHC, 5SrRNA or 39SrRNA promoters, or to the MIL gene C-terminal region. Binding by TPBF to the TPBF and MIL gene promoters was confirmed by in vitro assays. These results validate the in vitro model for TBP gene regulation and further suggest that TPBF may be autoregulated and may participate in the regulation of the MIL gene.
Transcriptional activation of the yeast HO gene involves the sequential action of DNA-binding and chromatin-modifying factors. Here we examine the role of the SAGA complex and the Nhp6 architectural transcription factor in HO regulation. Our data suggest that these factors regulate binding of the TATA-binding protein (TBP) to the promoter. A gcn5 mutation, eliminating the histone acetyltransferase present in SAGA, reduces the transcription of HO, but expression is restored in a gcn5 spt3 double mutant. We conclude that the major role of Gcn5 in HO activation is to overcome repression by Spt3. Spt3 is also part of SAGA, and thus two proteins in the same regulatory complex can have opposing roles in transcriptional regulation. Chromatin immunoprecipitation experiments show that TBP binding to HO is very weak in wild-type cells but markedly increased in an spt3 mutant, indicating that Spt3 reduces HO expression by inhibiting TBP binding. In contrast, it has been shown previously that Spt3 stimulates TBP binding to the GAL1 promoter as well as GAL1 expression, and thus, Spt3 regulates these promoters differently. We also find genetic interactions between TBP and either Gcn5 or the high-mobility-group protein Nhp6, including multicopy suppression and synthetic lethality. These results suggest that, while Spt3 acts to inhibit TBP interaction with the HO promoter, Gcn5 and Nhp6 act to promote TBP binding. The result of these interactions is to limit TBP binding and HO expression to a short period within the cell cycle. Furthermore, the synthetic lethality resulting from combining a gcn5 mutation with specific TBP point mutations can be suppressed by the overexpression of transcription factor IIA (TFIIA), suggesting that histone acetylation by Gcn5 can stimulate transcription by promoting the formation of a TBP/TFIIA complex.
TATA-binding protein (TBP) is an essential factor for eukaryotic transcription. In this study, we demonstrated a mouse cDNA encoding a 21 kDa TBP-like protein (TLP). The TLP ORF, carrying 186 amino acids, covered the entire 180 amino acids of the C-terminal conserved domain of mouse TBP with 39% identity and 76% similarity. Northern blot analysis demonstrated that TLP mRNAs were expressed in various mammalian tissues ubiquitously and that their distribution pattern was analogous to that of TBP. By using anti-TLP antibody, we demonstrated the existence of TLP proteins in various mammalian cells and tissues. The Drosophila TBP-related factor (TRF) is a neurogenesis-related transcription factor that binds to the TATA-box and activates transcription. TLP did not bind to the TATA-box nor direct transcription initiation. Multiple amino acids critical for TBP function were deleted or substituted in TLP, while amino acids in Drosophila TRF much resembled those in TBP. Similarity between Drosophila TRF and mouse TLP was considerably lower (alignment score 35) than that between Drosophila TBP and mouse TBP (alignment score 88). Identity of nucleotide sequences between mouse and putative human TLPs (94%) was higher than that between TBPs (91%) in these two animals. Expression of TLP was nearly constant throughout the P19 differentiation process. Accordingly, we suggest that, even if higher eukaryotes generally contain multiple tbp -related genes, TLP is not a bona fide mammalian counterpart of Drosophila TRF.
TATA-box-binding protein 2 (TBP2/TRF3) is a vertebrate-specific paralog of TBP that shares with TBP a highly conserved carboxy-terminal domain and the ability to bind the TATA box. TBP2 is highly expressed in oocytes whereas TBP is more abundant in embryos.
We find that TBP2 is proteolytically degraded upon meiotic maturation; after germinal vesicle breakdown relatively low levels of TBP2 expression persist. Furthermore, TBP2 localizes to the transcriptionally active loops of lampbrush chromosomes and is recruited to a number of injected promoters in oocyte nuclei. Using an altered binding specificity mutant reporter system we show that TBP2 promotes RNA polymerase II transcription in vivo. Intriguingly, TBP, which in oocytes is undetectable at the protein level, can functionally replace TBP2 when ectopically expressed in oocytes, showing that switching of initiation factors can be driven by changes in their expression. Proteolytic degradation of TBP2 is not required for repression of transcription during meiotic maturation, suggesting a redundant role in this repression or a role in initiation factor switching between oocytes and embryos.
The expression and transcriptional activity of TBP2 in oocytes show that TBP2 is the predominant initiation factor in oocytes, which is substituted by TBP on a subset of promoters in embryos as a result of proteolytic degradation of TBP2 during meiotic maturation.
TATA Binding Protein (TBP) is required for transcription initiation by all three eukaryotic RNA polymerases. It participates in transcriptional initiation at the majority of eukaryotic gene promoters, either by direct association to the TATA box upstream of the transcription start site or by indirectly localizing to the promoter through other proteins. TBP exists in solution in a dimeric form but binds to DNA as a monomer. Here, we present the first mathematical model for auto-catalytic TBP expression and use it to study the role of dimerization in maintaining the steady state TBP level.
We show that the autogenous regulation of TBP results in a system that is capable of exhibiting three steady states: an unstable low TBP state, one stable state corresponding to a physiological TBP concentration, and another stable steady state corresponding to unviable cells where no TBP is expressed. Our model predicts that a basal level of TBP is required to establish the transcription of the TBP gene, and hence for cell viability. It also predicts that, for the condition corresponding to a typical mammalian cell, the high-TBP state and cell viability is sensitive to variation in DNA binding strength. We use the model to explore the effect of the dimer in buffering the response to changes in TBP levels, and show that for some physiological conditions the dimer is not important in buffering against perturbations.
Results on the necessity of a minimum basal TBP level support the in vivo observations that TBP is maternally inherited, providing the small amount of TBP required to establish its ubiquitous expression. The model shows that the system is sensitive to variations in parameters indicating that it is vulnerable to mutations in TBP. A reduction in TBP-DNA binding constant can lead the system to a regime where the unviable state is the only steady state. Contrary to the current hypotheses, we show that under some physiological conditions the dimer is not very important in restoring the system to steady state. This model demonstrates the use of mathematical modelling to investigate system behaviour and generate hypotheses governing the dynamics of such nonlinear biological systems.
This article was reviewed by Tomasz Lipniacki, James Faeder and Anna Marciniak-Czochra.
Archaea (formerly archaebacteria) comprise a domain of life that is phylogenetically distinct from both Eucarya and Bacteria. Here we report the cloning of a gene from the Archaeon Sulfolobus shibatae that encodes a protein with strong homology to the TATA binding protein (TBP) of eukaryotes. Sulfolobus shibatae TBP is, however, almost as diverged from other archaeal TBPs that have been cloned as it is from eukaryotic TBPs. DNA binding studies indicate that S.shibatae TBP recognizes TATA-like A-box sequences that are present upstream of most archaeal genes. By quantitatively immunodepleting S.shibatae TBP from an in vitro transcription system, we demonstrate that Sulfolobus RNA polymerase is capable of transcribing the 16S/23S rRNA promoter weakly in the absence of TBP. Most significantly, we show that addition of recombinant S.shibatae TBP to this immunodepleted system leads to transcriptional stimulation and that this stimulation is dependent on the A-box sequence of the promoter. Taken together, these findings reveal fundamental similarities between the transcription machineries of Archaea and eukaryotes.
The recruitment of TATA box-binding protein (TBP) to promoters is one of the rate-limiting steps during transcription initiation. However, the global importance of TBP recruitment in determining the absolute and changing levels of transcription across the genome is not known. We used a genomic approach to explore the relationship between TBP recruitment to promoters and global gene expression profiles in Saccharomyces cerevisiae. Our data indicate that first, RNA polymerase III promoters are the most prominent binding targets of TBP in vivo. Second, the steady-state transcript levels of genes throughout the genome are proportional to the occupancy of their promoters by TBP, and changes in the expression levels of these genes are closely correlated with changes in TBP recruitment to their promoters. Third, a consensus TATA element does not appear to be a major determinant of either TBP binding or gene expression throughout the genome. Our results indicate that the recruitment of TBP to promoters in vivo is of universal importance in determining gene expression levels in yeast, regardless of the nature of the core promoter or the type of activator or repressor that may mediate changes in transcription. The primary data reported here are available at http://www.iyerlab.org/tbp.
In zebrafish, TATA-box-binding protein (TBP)-related factor 3, Trf3, is required for early development and initiation of hematopoiesis, and functions by promoting expression of a single target gene, mespa. Recent studies have shown that in murine muscle cells, TRF3 interacts with the TBP-associated factor TAF3. Here we investigate the role of Taf3 in zebrafish embryogenesis. We find that like Trf3-depleted zebrafish embryos, Taf3-depleted embryos exhibit multiple developmental defects and fail to undergo hematopoiesis. Both Trf3 and Taf3 are selectively bound to the mespa promoter and are required for mespa expression. Significantly, Taf3 interacts with Trf3 but not Tbp, and a Trf3 mutant that disrupts this interaction fails to support mespa transcription, early development and hematopoiesis. Thus, a selective interaction between Trf3 and Taf3 is required for early zebrafish development and initiation of hematopoiesis. Finally, we provide evidence that TRF3 and TAF3 are also required for hematopoiesis initiation in the mouse.
Taf3; Trf3; zebrafish; hematopoiesis; embryogenesis
Using a genetic screen, we isolated three TATA-binding protein (TBP) mutants that increase transcription from promoters that are repressed by the Cyc8-Tup1 or Sin3-Rpd3 corepressors or that lack an enhancer element, but not from an equivalently weak promoter with a mutated TATA element. Increased transcription is observed when the TBP mutants are expressed at low levels in the presence of wild-type TBP. These TBP mutants are unable to support cell viability, and they are toxic in strains lacking Rpd3 histone deacetylase or when expressed at higher levels. Although these mutants do not detectably bind TATA elements in vitro, genetic and chromatin immunoprecipitation experiments indicate that they act directly at promoters and do not increase transcription by titration of a negative regulatory factor(s). The TBP mutants are mildly defective for associating with promoters responding to moderate or strong activators; in addition, they are severely defective for RNA polymerase (Pol) III but not Pol I transcription. These results suggest that, with respect to Pol II transcription, the TBP mutants specifically increase expression from core promoters. Biochemical analysis indicates that the TBP mutants are unaffected for TFIID complex formation, dimerization, and interactions with either the general negative regulator NC2 or the N-terminal inhibitory domain of TAF130. We speculate that these TBP mutants have an unusual structure that allows them to preferentially access TATA elements in chromatin templates. These TBP mutants define a criterion by which promoters repressed by Cyc8-Tup1 or Sin3-Rpd3 resemble enhancerless, but not TATA-defective, promoters; hence, they support the idea that these corepressors inhibit the function of activator proteins rather than the Pol II machinery.
The retinoblastoma protein (RB) represses RNA polymerase III transcription effectively both in vivo and in vitro. Here we demonstrate that the general transcription factors snRNA-activating protein complex (SNAPc) and TATA binding protein (TBP) are important for RB repression of human U6 snRNA gene transcription by RNA polymerase III. RB is associated with SNAPc as detected by both coimmunoprecipitation of endogenous RB with SNAPc and cofractionation of RB and SNAPc during chromatographic purification. RB also interacts with two SNAPc subunits, SNAP43 and SNAP50. TBP or a combination of TBP and SNAPc restores efficient U6 transcription from RB-treated extracts, indicating that TBP is also involved in RB regulation. In contrast, the TBP-containing complex TFIIIB restores adenovirus VAI but not human U6 transcription in RB-treated extracts, suggesting that TFIIIB is important for RB regulation of tRNA-like genes. These results suggest that different classes of RNA polymerase III-transcribed genes have distinct general transcription factor requirements for repression by RB.
Candida albicans is the major fungal pathogen in humans, yet little is known about transcriptional regulation in this organism. Therefore, we have isolated, characterized, and expressed the C. albicans TATA-binding protein (TBP) gene (TBP1), because this general transcription initiation factor plays a key role in the activation and regulation of eukaryotic promoters. Southern and Northern blot analyses suggest that a single C. albicans TBP1 locus is expressed at similar levels in the yeast and hyphal forms of this fungus. The TBP1 open reading frame is 716 bp long and encodes a functional TBP of 27 kDa. C. albicans TBP is capable of binding specifically to a TATA box in vitro, substituting for the human TBP to activate basal transcription in vitro, and suppressing the lethal Δspt15 mutation in Saccharomyces cerevisiae. The predicted amino acid sequences of TBPs from C. albicans and other organisms reveal a striking pattern of C-terminal conservation and N-terminal variability: the C-terminal DNA-binding domain displays at least 80% amino acid sequence identity to TBPs from fungi, flies, nematodes, slime molds, plants, and humans. Sequence differences between human and fungal TPBs in the DNA-binding domain may represent potential targets for antifungal therapy.
We have analysed the function of sequences in the TATA/initiator region of the promoter of Ea, a class II gene of the Major Histocompatibility Complex. We find that the Ea promoter contains an initiator element with a strong influence on transcription. We also find that the Ea promoter does contain a bona fide TATA box, which can be recognized by the TATA binding protein (TBP), and that TBP is required for transcriptional activity. For activity, TBP must be included within a larger TFIID complex, as Ea transcription in a heat-treated extract can be restored by immunopurified TFIID but not by TBP alone. On the other hand, the TATA motif can be eliminated without significantly affecting either the efficiency or the startsites of transcription. This suggests that TBP, even in this TATA-containing promoter, is held in place by other components of the initiation complex, regardless of its affinity for the underlying DNA.
The TATA sequence of the human, estrogen-responsive pS2 promoter is complexed in vivo with a rotationally and translationally positioned nucleosome (NUC T). Using a chromatin immunoprecipitation assay, we demonstrate that TATA binding protein (TBP) does not detectably interact with this genomic binding site in MCF-7 cells in the absence of transcriptional stimuli. Estrogen stimulation of these cells results in hyperacetylation of both histones H3 and H4 within the pS2 chromatin encompassing NUC T and the TATA sequence. Concurrently, TBP becomes associated with the pS2 promoter region. The relationship between histone hyperacetylation and the binding of TBP was assayed in vitro using an in vivo-assembled nucleosomal array over the pS2 promoter. With chromatin in its basal state, the binding of TBP to the pS2 TATA sequence at the edge of NUC T was severely restricted, consistent with our in vivo data. Acetylation of the core histones facilitated the binding of TBP to this nucleosomal TATA sequence. Therefore, we demonstrate that one specific, functional consequence of induced histone acetylation at a native promoter is the alleviation of nucleosome-mediated repression of the binding of TBP. Our data support a fundamental role for histone acetylation at genomic promoters in transcriptional activation by nuclear receptors and provide a general mechanism for rapid and reversible transcriptional activation from a chromatin template.
The parasites Leishmania spp., Trypanosoma brucei, and Trypanosoma cruzi are the trypanosomatid protozoa that cause the deadly human diseases leishmaniasis, African sleeping sickness, and Chagas disease, respectively. These organisms possess unique mechanisms for gene expression such as constitutive polycistronic transcription of protein-coding genes and trans-splicing. Little is known about either the DNA sequences or the proteins that are involved in the initiation and termination of transcription in trypanosomatids. In silico analyses of the genome databases of these parasites led to the identification of a small number of proteins involved in gene expression. However, functional studies have revealed that trypanosomatids have more general transcription factors than originally estimated. Many posttranslational histone modifications, histone variants, and chromatin modifying enzymes have been identified in trypanosomatids, and recent genome-wide studies showed that epigenetic regulation might play a very important role in gene expression in this group of parasites. Here, we review and comment on the most recent findings related to transcription initiation and termination in trypanosomatid protozoa.
The interaction of yeast TFIIIB with the region upstream of the SUP4 tRNATyr gene was extensively probed by use of photoreactive phosphodiesters, deoxyuridines, and deoxycytidines that are site specifically incorporated into DNA. The TATA binding protein (TBP) was found to be in close proximity to the minor groove of a TATA-like DNA sequence that starts 30 nucleotides upstream of the start site of transcription. TBP was cross-linked to the phosphate backbone of DNA from bp −30 to −20 in the nontranscribed strand and from bp −28 to −24 in the transcribed strand (+1 denotes the start site of transcription). Most of the major groove of DNA in this region was shown not to be in close proximity to TBP, thus resembling the binding of TBP to the TATA box, with one notable exception. TBP was shown to interact with the major groove of DNA primarily at bp −23 and to a lesser degree at bp −25 in the transcribed strand. The stable interaction of TBP with the major groove at bp −23 was shown to require the B" subunit of TFIIIB. The S4 helix and flanking region of TBP were shown to be proximal to the major groove of DNA by peptide mapping of the region of TBP cross-linked at bp −23. Thus, TBP in the TFIIIB-SUP4 gene promoter region is bound in the same direction as TBP bound to the TATA box with respect to the transcription start site. The B" and TFIIB-related factor (BRF) subunits of TFIIIB are positioned on opposite sides of the TBP-DNA core of the TFIIIB complex, as indicated by correlation of cross-linking data to the crystal structure of the TBP-TATA box complex. Evidence is given for BRF binding near the C-terminal stirrup of TBP, similar to that of TFIIB near the TBP-TATA box complex. The protein clamp formed around the TBP-DNA complex by BRF and B" would help explain the long half-life of the TFIIIB-DNA complex and its resistance to polyanions and high salt. The path of DNA traversing the surface of TBP at the 3′ end of the TATA-like element in the SUP4 tRNA gene is not the same as that of TBP bound to a TATA box element, as shown by the cross-linking of TBP at bp −23.
We attempted to devise a transcription system in which a particular
DNA sequence of interest could be inducibly expressed under the
control of a modified polymerase III (pol III) promoter. Its activation requires
a mutated transcription factor not contained endogenously in human
cells. We constructed such a promoter by fusing elements of the β-lactamase gene of Escherichia
coli, containing a modified TATA-box and a pol III terminator,
to the initiation region of the human U6 gene. This construct functionally
resembles a 5′-regulated pol III gene
and its transcribed segment can be exchanged for an arbitrary sequence.
Its transcription in vitro by pol III requires the
same factors as the U6 gene with the major exception that the modified
TATA-box of this construct only interacts with a TATA-binding protein (TBP)
mutant (TBP-DR2) but not with TBP wild-type (TBPwt). Its transcription
therefore requires TBP-DR2 exclusively instead of TBPwt. In order
to render the system inducible, we fused the gene coding for TBP-DR2
to a tetracycline control element and stably transfected this new
construct into HeLa cells. Induction of such a stable and viable
clone with tetracycline resulted in the expression of functional
TBP-DR2. This system may conceptually be used in the future to inducibly
express an arbitrary DNA sequence in vivo under
the control of the above mentioned promoter.
Interaction of the TATA box-binding protein (TBP) with promoters of RNA polymerase II-transcribed genes is an early and essential step in mRNA synthesis. Previous studies have demonstrated that the rate-limiting binding of TBP to a TATA element can be influenced by transcriptional regulatory proteins. To identify additional factors that may regulate DNA binding by TBP in vivo, we performed a genetic selection for extragenic suppressors of a yeast TBP mutant that exhibits altered and relaxed DNA binding specificity. This analysis has led to the discovery of a previously unidentified gene, RTF1. The original rtf1 suppressor mutation, which encodes a single amino acid change in Rtf1, and an rtf1 null allele suppress the effects of the TBP specificity mutant by altering transcription initiation. Differences in the patterns of transcription initiation in these strains strongly suggest that the rtf1 missense mutation is distinct from a simple loss-of-function allele. The results of genetic crosses indicate that suppression of TBP mutants by mutations in RTF1 occurs in an allele-specific fashion. In a strain containing wild-type TBP, the rtf1 null mutation suppresses the transcriptional effects of a Ty delta insertion mutation in the promoter of the HIS4 gene, a phenotype also conferred by the TBP altered-specificity mutant. Finally, as shown by indirect immunofluorescence experiments, Rtf1 is a nuclear protein. Taken together, our findings suggest that Rtf1 either directly or indirectly regulates the DNA binding properties of TBP and, consequently, the relative activities of different TATA elements in vivo.
The TATA binding protein (TBP) is an essential transcription initiation factor in Archaea and Eucarya. Bacteria lack TBP, and instead use sigma factors for transcription initiation. TBP has a symmetric structure comprising two repeated TBP domains. Using sequence, structural and phylogenetic analyses, we examine the distribution and evolutionary history of the TBP domain, a member of the helix-grip fold family. Our analyses reveal a broader distribution than for TBP, with TBP-domains being present across all three domains of life. In contrast to TBP, all other characterized examples of the TBP domain are present as single copies, primarily within multidomain proteins. The presence of the TBP domain in the ubiquitous DNA glycosylases suggests that this fold traces back to the ancestor of all three domains of life. The TBP domain is also found in RNase HIII, and phylogenetic analyses show that RNase HIII has evolved from bacterial RNase HII via TBP-domain fusion. Finally, our comparative genomic screens confirm and extend earlier reports of proteins consisting of a single TBP domain among some Archaea. These monopartite TBP-domain proteins suggest that this domain is functional in its own right, and that the TBP domain could have first evolved as an independent protein, which was later recruited in different contexts.
The complexity of the core promoter transcription machinery has emerged as an additional level of transcription regulation that is used during vertebrate development. Recent studies, including one published in BMC Biology, provide mechanistic insights into how the TATA binding protein (TBP) and its vertebrate-specific paralog TBP2 (TRF3) switch function during the transition from the oocyte to the embryo.
See research article http://www.biomedcentral.com/1741-7007/7/45
The post-transcriptional control of gene expression is becoming increasingly important in the understanding of regulated events in eukaryotic cells. The parasitic kinetoplastids have a unique reliance on such processes, because their genome is organized into polycistronic transcription units in which adjacent genes are not coordinately regulated. Indeed, the number of RNA-binding proteins predicted to be encoded in the genome of kinetoplastids is unusually large, invoking the presence of unique RNA regulators dedicated to gene expression in these evolutionarily ancient organisms. Here, we report that a small CCCH zinc finger protein, TbZFP3, enhances development between life-cycle stages in Trypanosoma brucei. Moreover, we demonstrate that this protein interacts both with the translational machinery and with other small CCCH proteins previously implicated in trypanosome developmental control. Antibodies to this protein also co-immunoprecipitate EP procyclin mRNA and encode the major surface antigen of insect forms of T. brucei. Strikingly, although TbZFP3 is constitutively expressed, it exhibits developmentally regulated association with polyribosomes, and mutational analysis demonstrates that this association is essential for the expression of phenotype. TbZFP3 is therefore a novel regulator of developmental events in kinetoplastids that acts at the level of the post-transcriptional control of gene expression.