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PubMed1997

Nucleotide sequence of a ubiquitin-extension protein gene in Nicotiana tabacum and its expression pattern upon heat shock.

Using a tobacco cDNA clone as a probe, a genomic clone coding for a tobacco ubiquitin extension protein was isolated. Nucleotide sequence determination of the clone identified an open reading frame for an ubiquitin unit of 76 amino acids and an extension tail of 80 amino acids. To the 5' upstream of the open reading frame, a sequence of 1,110 nucleotides was determined and a putative CCAAT box and TATA box were located. An intron sequence was not apparent as the open reading frame for the ubiquitin-extension protein was continuous for 468 nucleotides. In the 3' downstream region of the gene, a putative polyadenylation signal and GU rich region were observed. Northern blot analysis of tobacco transcripts with the tail part of the gene as a probe specifically identified the 1.3 kb transcript. The Northern signal at 1.3 kb indicated that the expression of the ubiquitin-extension protein gene in tobacco is downregulated upon heat-shock.

Amino Acid Sequence↗
PubMed1995

Characterization of a novel transcript of retroviral origin expressed in rat heart and liver.

Eleven cDNA clones of retroviral origin (SORO) have been isolated and characterized from a rat cardiac cDNA library that are highly homologous to the RAL-element family of rat retroviral endogenous sequences. SORO-1, the largest of the clones isolated at approximately 3.5 kb, contains a characteristic long terminal repeat (LTR) that is unique to previously identified retroposons. Its LTR contains elements homologous to the CAAT box, a TATA box, a core enhancer sequence, and potential binding sites for GATA. SORO-1 hybridizes to a mRNA of approximately 7 kb, that is present both in rat heart and liver. This transcript was not, however, detected in other tissues (fast and slow skeletal muscles, brain, kidney, testis, lung, intestine, spleen) or from any other species (man, monkey, mouse, dog, rabbit, chicken, cow, yeast) examined. During postnatal cardiac development, the expression of the transcript appears to be down-regulated and is present at levels 10-fold greater in neonates than in adults. The function of this retroposon that is expressed in rat heart and liver has not yet been determined, but the sequence analysis of its LTR and its expression pattern suggest that it may be regulated by specific trans-acting factors that are present in both rat heart and liver.

Animals↗
PubMed1996

Three-exon structure of the gene encoding the rat prion protein and its expression in tissues.

The prion protein (PrP), encoded by a chromosomal gene, is associated with development of the neurodegeneration of prion-induced diseases. Since determination of the complete structure of the gene encoding PrP is important for understanding gene expression in the central nervous system (CNS), the nucleotide (nt) sequence of the isolated whole gene encoding rat PrP (raPrP) was determined. The rat PrP gene (raPrP) spans 16 kilobases (kb) of the rat genome and contains three exons of 19-47 base pairs (bp), 98 bp, and 2 kb separated by two introns of 2.2 kb and 11 kb. The first and second exons are noncoding, while the third exon contains a short 5' untranslated region, the entire 762-bp open reading frame (ORF), and a 3' untranslated region. The putative raPrP promoter in the 5' flanking region contains putative Sp1, AP-1, and AP-2 binding sites without a consensus TATA box. This TATA box-deficient feature, coupled with the presence of a high G+C content and Sp1-binding sites in the raPrP promoter, characterizes it as a housekeeping gene. Analysis of the raPrP cDNA 5'-end showed that raPrP mRNA transcription was initiated at multiple sites. Northern blot analysis showed that the levels of raPrP mRNA varied among rat tissues, with the highest levels found in the brain and placenta. This determination of raPrP nt sequences, including the introns and the 5' and 3' flanking regions, may make it possible to elucidate cis-acting elements that regulate the expression of this gene in different tissues and cell lines.

Amino Acid Sequence↗
PubMed2000

[The function of T7 promoter as cis-acting elements for polymerase II in eukaryotic cell].

Using the chlorampheniol acetyltransterase gene as reporter, the function of phage T7 promoter in mammalian cells was studied by inhibition of transcription with alpha-amanitin. The experiment proved that the reporter under T7 promoter was transcribed by RNA polymerase II. Competitive electropho retic mobility shift assay (CEMSA) with TATA box, CAAT box, GC box and octamer showed that the TATA box was competitive molecular for synthetic T7 promoter. It is possible that T7 promoter is bound with TF II D transcription factor. The TATA box and octamer were inserted into Pvu II site upstream from the T7 promoter of pT7CAT. Two recombinant plasmids, pT7TATACAT and pT7OCTCAT, were constructed and transfected into CHO cells. CAT-activity test showed that T7 promoter strength was increased by octamer factor, not by TATA box. These results suggested that T7 promoter functions as cis-acting elements of RNA polymerase II transcriptional system in eucaryotic cells.

Bacteriophage T7↗
PubMed1989

The MF alpha 1 gene of Saccharomyces cerevisiae: genetic mapping and mutational analysis of promoter elements.

The activity and cell-type specificity of the promoter of the MF alpha 1 gene of Saccharomyces cerevisiae were examined by measuring expression of an MF alpha 1-SUC2 gene fusion in MATa, MAT alpha, and MATa/MAT alpha cells. A high level of invertase activity was observed only in MAT alpha cells. Weak expression occurred in MATa cells when the hybrid gene was carried on a multicopy plasmid or on a centromere-containing plasmid, but not when the hybrid gene was integrated at the normal MF alpha 1 locus. Analysis of a set of 5'-deletions of the promoter region of the MF alpha 1-SUC2 gene on the multicopy plasmid indicated that sequences from -354 to -274 upstream of the translational start site were required for high level expression in MAT alpha cells. Smaller internal deletions and insertions within the promoter region of the MF alpha 1-SUC2 gene were inserted into the genome at the normal MF alpha 1 locus. These mutations further delineated four promoter domains important for expression: (1) two 26 bp elements (-365 to -340 and -312 to -287) with imperfect dyad symmetry; (2) a 40 bp segment (-264 to -226) that lies about 120 bp upstream of the TATA box; and (3) the TATA box itself (-128 to -122). The transcriptional start sites of the normal MF alpha 1 promoter and of a mutant lacking the TATA box were determined. The MF alpha 1 locus was mapped to the left arm of chromosome XVI, about 22 cM centromere-proximal to the PEP4 gene.

Base Sequence↗
PubMed1994

2.1 A resolution refined structure of a TATA box-binding protein (TBP).

The three-dimensional structure of a TATA box-binding protein (TBP2) from Arabidopsis thaliana has been refined at 2.1 A resolution. TBPs are general eukaryotic transcription factors that participate in initiation of RNA synthesis by all three eukaryotic RNA polymerases. The carboxy-terminal portion of TBP is a unique DNA-binding motif/protein fold, adopting a highly symmetric alpha/beta structure that resembles a molecular saddle with two stirrup-like loops. A ten-stranded, antiparallel beta-sheet provides a concave surface for recognizing class II nuclear gene promoters, while the four amphipathic alpha-helices on the convex surface are available for interaction with other transcription factors. The myriad interactions of TBP2 with components of the transcription machinery are discussed.

Amino Acid Sequence↗
PubMed2002

Evidence that TAF-TATA box-binding protein interactions are required for activated transcription in mammalian cells.

Surfaces of human TATA box-binding protein (hsTBP) required for activated transcription in vivo were defined by constructing a library of surface residue substitution mutations and assaying them for their ability to support activated transcription in transient-transfection assays. In earlier work, three regions were identified where mutations inhibited activated transcription without interfering with TATA box DNA binding. One region is on the upstream surface of the N-terminal TBP repeat with respect to the direction of transcription and corresponds to the TBP surface that interacts with TFIIA. A second region on the stirrup of the C-terminal TBP repeat corresponds to the TFIIB-binding surface. Here we report that the third region where mutations inhibit activated transcription in mammalian cells, the convex surface of the N-terminal repeat, corresponds to a surface on TBP that interacts with hsTAF1, the major scaffold subunit of TFIID. Since mutations at the center of the hsTAF1-interacting region inhibit the ability of the protein to support activated transcription in vivo, these results are consistent with the conclusion that an interaction between hsTBP and TAF(II)s is required for activated transcription in mammalian cells.

Amino Acid Sequence↗
PubMed1988

Unusual DNA structure in the regulatory region of the human papovavirus JC virus.

The human papovavirus JC virus (JCV) was analyzed for the presence of unusual DNA conformations. Recombinant plasmids containing 60% of the JCV prototype Mad-1 strain DNA were constructed and analyzed with both enzymatic and chemical probes. Fine-mapping studies revealed that the most prominent S1 nuclease-sensitive and bromoacetaldehyde-modified sites were located within the TATA boxes of each 98-base-pair tandem repeat. Further studies revealed that the S1 nuclease-sensitive site in the first TATA box (proximal to the origin) was approximately 50-fold stronger than the site in the second TATA box (distal from the origin). Deletion of the first TATA box drastically reduced the extent of bromoacetaldehyde modification in the second TATA box, whereas deletion of the second TATA box had little or no effect on the reactivity at the first TATA box. Hence, the biological and conformational role of the second TATA box remains unclear. No supercoil-induced relaxation was found, and reactions with the probes were not pH dependent. Also, fragments containing this regulatory region did not appear to be bent, although the A+T-rich segment contained a tract of eight consecutive A's. We conclude that the regulatory region of JCV contains non-B, but right-handed, DNA conformations which account for this behavior.

BK Virus↗
PubMed1999

An element with palindromic structure is required for the expression of TBP (TATA box-binding protein) gene in Drosophila melanogaster.

Previously we showed that the 5'-flanking regions between -261 and -207 of the Drosophila melanogaster TBP (TATA box binding protein) gene is important for its expression. We further made serial deletion mutants in this region and analyzed their promoter activities using the transient transfection assay. We found that the 16 bp deletion from -261 to -245 greatly reduces the promoter activity of the Drosophila TBP gene. The 16 bp DNA element contains half of a 11 bp long palindromic sequence, CTTTT-GAAAAG. Disruption of the palindromic sequence by site-directed mutagenesis severely affected promoter activity. In addition, the electrophoretic mobility shift assay showed that the oligonucleotide containing the palindromic sequence can make specific DNA/protein complexes when it was mixed with the Drosophila nuclear extract, suggesting that it interacts with nuclear protein(s). Our data suggest that the palindromic sequence has a critical role in the expression of the Drosophila TBP gene.

Animals↗
PubMed2000

Binding mechanisms of TATA box-binding proteins: DNA kinking is stabilized by specific hydrogen bonds.

One of the common mechanisms of DNA bending by minor groove-binding proteins is the insertion of protein side chains between basepair steps, exemplified in TBP (TATA box-binding protein)/DNA complexes. At the central basepair step of the TATA box TBP produces a noticeable decrease in twist and an increase in roll, while engaging in hydrogen bonds with the bases and sugars. This suggests a mechanism for the stabilization of DNA kinks that was explored here with ab initio quantum mechanical calculations and molecular dynamics/potential of mean force calculations. The hydrogen bonds are found to contribute the energy necessary to drive the conformational transition at the central basepair step. The Asn, Thr, and Gly residues involved in hydrogen bonding to the DNA bases and sugar oxygens form a relatively rigid motif in TBP. The interaction of this motif with DNA is found to be responsible for inducing the untwisting and rolling of the central basepair step. Notably, direct readout is shown not to be capable of discriminating between AA and AT steps, as the strength of the hydrogen bonds between TBP and the DNA are the same for both sequences. Rather, the calculated free energy cost for an equivalent conformational transition is found to be sequence-dependent, and is calculated to be higher for AA steps than for AT steps.

Biophysical Phenomena↗
PubMed2003

Transcription mapping of human herpesvirus 8 genes encoding viral interferon regulatory factors.

The human herpesvirus 8 (HHV-8) genome contains four tandemly arranged genes encoding viral interferon regulatory factors (vIRF-1 to 4) located between genes 57 and 58. Transcript mapping techniques were employed to determine the sizes, ends and splicing patterns of mRNAs specified by these genes in HHV-8-infected cell lines untreated or chemically induced into the lytic growth cycle. Depending on the cell line used, vIRF-3 transcription was minimally or not induced (i.e. expressed with latent kinetics), whereas the other vIRFs were inducible (i.e. expressed with lytic kinetics). Each gene possessed its own promoter (or promoters) and polyadenylation sites, and all but vIRF-1 were spliced from two exons. vIRF-1 was transcribed in uninduced and induced cells from a single initiation site preceded by a TATA box, with the possible use of an additional TATA box and initiation site in uninduced cells. In induced cells, vIRF-2 was transcribed from a single major initiation site preceded by a TATA box, and vIRF-4 was expressed from two sites each preceded by a TATA box. Transcripts for these genes were insufficiently abundant in uninduced cells to map the 5'-ends. vIRF-3 lacks an obvious TATA box and exhibited heterogeneous 5'-ends in uninduced and induced cells. These data clarify and extend our understanding of the structure and transcription of the HHV-8 vIRF genes.

Amino Acid Sequence↗
PubMed1999

TATA box-binding protein (TBP)-related factor 2 (TRF2), a third member of the TBP family.

The TATA box-binding protein (TBP) is an essential component of the RNA polymerase II transcription apparatus in eukaryotic cells. Until recently, it was thought that the general transcriptional machinery was largely invariant and relied on a single TBP, whereas a large and diverse collection of activators and repressors were primarily responsible for imparting specificity to transcription initiation. However, it now appears that the "basal" transcriptional machinery also contributes to specificity via tissue-specific versions of TBP-associated factors as well as a tissue-specific TBP-related factor (TRF1) responsible for gene selectivity in Drosophila. Here we report the cloning of a TBP-related factor (TRF2) that is found in humans, Drosophila, Caenorhabditis elegans, and other metazoans. Like TRF1 and TBP, TRF2 binds transcription factor IIA (TFIIA) and TFIIB and appears to be part of a larger protein complex. TRF2's primary amino acid structure suggests divergence in the putative DNA binding domain, and not surprisingly, it fails to bind to DNA containing canonical TATA boxes. Most importantly, TRF2 is associated with loci on Drosophila chromosomes distinct from either TBP or TRF1, so it may have different promoter specificity and regulate a select subset of genes. These findings suggest that metazoans have evolved multiple TBPs to accommodate the vast increase in genes and expression patterns during development and cellular differentiation.

Amino Acid Sequence↗
PubMed1994

Topological localization of the human transcription factors IIA, IIB, TATA box-binding protein, and RNA polymerase II-associated protein 30 on a class II promoter.

The human general transcription factors IIA and IIB bind directly to the TATA box-binding protein (TBP), and modulate transcription initiation by RNA polymerase II. RAP30, the small subunit of TFIIF, binds to TFIIB and RNA polymerase II and recruits RNA polymerase II to a preinitiation complex containing TBP and TFIIB. By using the adenovirus 2 major late promoter tagged site-specifically with the photoactivatible cross-linking reagent N3R-dUMP we have localized TBP, two subunits of TFIIA (A35 and A21), TFIIB, and RAP30 along promoter DNA. TFIIA cross-linked to the coding strand opposite TBP at the TATA box and cross-linked upstream of TBP around position -40. RAP30 cross-linked strongly and TFIIB weakly to the coding strand just downstream of TBP at -19. We interpret these data in the context of a molecular structure for the TBP promoter complex.

Adenoviridae↗
PubMed1994

Equivalent mutations in the two repeats of yeast TATA-binding protein confer distinct TATA recognition specificities.

To investigate the process of TATA box recognition by the TATA box-binding protein (TBP), we have performed a detailed genetic and biochemical analysis of two Saccharomyces cerevisiae TBP mutants with altered DNA-binding specificity. The mutant proteins have amino acid substitutions (Leu-205 to Phe and Leu-114 to Phe) at equivalent positions within the two repeats of TBP that are involved in TATA element binding. In an in vivo assay that employs a nearly complete set of single point mutations of the consensus TATAAA sequence, one of the TBP mutants (TBP-L114F) recognizes the sequence TATAAG, while the other TBP mutant (TBP-L205F) recognizes one substitution at the first position of the TATA element, CATAAA, and three substitutions at the 3' end of the TATA box. Specificity patterns determined from in vitro transcription experiments with purified recombinant wild-type TBP and TBP-L205F agree closely with those observed in vivo, indicating that altered TATA utilization in the mutant strains is a direct consequence of altered TATA recognition by the mutant TBPs. The distinct TATA recognition patterns exhibited by TBP-L114F and TBP-L205F strongly suggest that in vivo, TBP binds to the TATA element in a specific orientation. The orientation predicted from these studies is further supported by the identification of intragenic suppressors that correct the defect of TBP-L205F. This orientation is consistent with that observed in vitro by crystallographic analyses of TBP-TATA box complexes. Finally, the importance of altered DNA-binding specificity in transcriptional regulation at the S. cerevisiae his4-912 delta promoter was addressed for TBP-L205F. A mutational analysis of this promoter region demonstrates that the nonconsensus TATA element CATAAA is required for a transcriptional effect of TBP-L205F in vivo. This finding suggests that the interaction of TBP with nonconsensus TATA elements may play an important regulatory role in transcription initiation.

Base Sequence↗
PubMed1999

Analysis of the latency-associated transcript/UL1-3.5 gene cluster promoter complex of pseudorabies virus.

During latency, pseudorabies virus (PRV) DNA is preferentially retained in the neurons of the trigeminal ganglion and a spliced 8.5-kilobase poly-A RNA, designated large latency transcript (LLT), is synthesized. Because LLT is the only transcript made during the latent phase, the LLT promoter may be unique among all other PRV promoters that are active in productive infections. Organization of the PRV LLT promoter is quite complex because it coincides with the UL1-3.5 gene cluster promoter, but in the opposite orientation. By conventional designation, LLT is transcribed in the rightward direction while the UL1-3.5 gene cluster is transcribed in the leftward orientation. In this work, activities of the LLT promoter and the UL1-3.5 gene cluster promoter were investigated by transient reporter gene expression assay in cells of neuronal and non-neuronal origins. There are two TATA boxes in this region. We examined the promoter activities of the first TATA box with its 5' sequence (LAP1) and the second TATA box with its 5' sequence (LAP2). The UL1-3.5 promoter driven constructs gave no reporter gene activity in any of the experiments. Reporter gene activity was detected with LAP2 gene constructs, but not with LAP1 constructs, in both neuronal and non-neuronal cells. This is surprising because transcription of PRV LLT in vivo has been attributed to LAP1 and the initiation site was mapped downstream of the LAP1 TATA box and upstream of the LAP2 TATA box. Although LAP1 was not active in these experiments, there was a 3- to 10-fold enhancement of activity when LAP1 and LAP2 were placed in tandem.

Animals↗
PubMed2001

A novel upstream RNA polymerase III promoter element becomes essential when the chromatin structure of the yeast U6 RNA gene is altered.

The Saccharomyces cerevisiae U6 RNA gene, SNR6, possesses upstream sequences that allow productive binding in vitro of the RNA polymerase III (Pol III) transcription initiation factor IIIB (TFIIIB) in the absence of TFIIIC or other assembly factors. TFIIIC-independent transcription of SNR6 in vitro is highly sensitive to point mutations in a consensus TATA box at position -30. In contrast, the TATA box is dispensable for SNR6 transcription in vivo, apparently because TFIIIC bound to the intragenic A block and downstream B block can recruit TFIIIB via protein-protein interactions. A mutant allele of SNR6 with decreased spacing between the A and B blocks, snr6-Delta42, exhibits increased dependence on the upstream sequences in vivo. Unexpectedly, we find that in vivo expression of snr6-Delta42 is much more sensitive to mutations in a (dT-dA)(7) tract between the TATA box and transcription start site than to mutations in the TATA box itself. Inversion of single base pairs in the center of the dT-dA tract nearly abolishes transcription of snr6-Delta42, yet inversion of all 7 base pairs has little effect on expression, indicating that the dA-dT tract is relatively orientation independent. Although it is within the TFIIIB footprint, point mutations in the dT-dA tract do not inhibit TFIIIB binding or TFIIIC-independent transcription of SNR6 in vitro. In the absence of the chromatin architectural protein Nhp6, dT-dA tract mutations are lethal even when A-to-B block spacing is wild type. We conclude that the (dT-dA)(7) tract and Nhp6 cooperate to direct productive transcription complex assembly on SNR6 in vivo.

AT Rich Sequence↗
PubMed1990

Two different RNA polymerase II initiation complexes can assemble on the rat brain creatine kinase promoter.

The rat brain creatine kinase (CKB) gene has a structurally complex promoter. Although it contains a consensus RNA polymerase II TATA box, this is located at -60 relative to the main transcription start site, an unprecedented distance for mammalian TATA boxes. Steady-state messenger RNA for CKB is extremely abundant in brain, yet barely detectable in liver. Using a nuclear run-on analysis, we show that this difference is due, at least in part, to a difference in the rate at which CKB is transcribed in these tissues. We also demonstrate a tissue-specific hypersensitive site in the promoter region of the CKB gene. In vitro transcription experiments using nuclear extract isolated from liver, brain, and HeLa cells show a marked difference in the way these extracts recognize the CKB promoter. Two different preinitiation complexes can assemble on the CKB promoter, one which initiated at the same downstream start-site used in normal adult rat brain and one which initiated 35 base pairs further upstream. Extracts from brain form exclusively the downstream initiating complex. However, liver extracts favor formation of the upstream initiating complex, but formation of this complex can be blocked if brain or HeLa extract is added first. We demonstrate that the upstream consensus TATA box can function as a TATA box to mediate upstream initiation, but is not required for downstream initiation. We have shown previously that brain and HeLa cell nuclear extracts contain an abundant factor, TARP, which binds to the upstream consensus TATA box and have proposed that this factor is distinct from the classical TATA box binding factor TFIID. We propose that binding of TARP to the upstream consensus TATA box may block formation of the upstream initiating complex.

Animals↗
PubMed1994

Direct recognition of initiator elements by a component of the transcription factor IID complex.

A core promoter element called an initiator (Inr) overlaps the transcription start site of numerous mammalian protein-coding genes. In promoters that lack a TATA box, the Inr is functionally analogous to TATA, in that it is capable of directing basal transcription by RNA polymerase II and of determining the precise site of transcription initiation. In promoters that contain a TATA box, the Inr can greatly enhance promoter strength. Mammalian Inr consensus sequences have been defined through functional studies and sequence comparisons of the start site regions of protein-coding genes. Here, we show that, in a DNase I footprinting assay with synthetic promoters, the purified TATA-binding protein complex TFIID specifically contacted the Inr. The TFIID-Inr interaction relies on the precise nucleotides needed for Inr function. Detection of the interaction was dependent either on a TATA box or on Sp1 bound to upstream sites. Furthermore, recombinant TFIIB appeared to influence the TFIID-Inr interaction, whereas TFIIA stabilized the TFIID-TATA interaction. These results demonstrate that distinct components of TFIID interact with the TATA boxes and Inr elements of core promoters for RNA polymerase II.

Animals↗