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PubMed1992

TATA box-independent transcription of the human tissue plasminogen activator gene initiates within a sequence conserved in related genes.

Transcription of the human tissue-type plasminogen activator (tPA) gene has been reported to initiate from a single site proximal to a TATA box motif [1985, J. Biol. Chem. 260, 11223-11230]. In this study, we utilized primer extension analysis to evaluate the tPA mRNA start site in phorbol-12-myristate 13-acetate (PMA) induced WI-38 human lung fibroblast cells. Whilst some tPA mRNA initiated from the predicted TATA-proximal location (+1), a 10-fold greater proportion of tPA mRNA transcripts initiated 110 bases downstream from a sequence conserved and utilized as the TATA-independent transcription start site in the rodent tPA genes. Moreover, the transfection and expression in different cell types of a cosmid containing the entire human tPA gene resulted in utilization of the same downstream (+110) start site. We propose that this, rather than the previously published position, is the major transcriptional initiation point for the human tPA gene. A core sequence (5'-CAGAGCTG-3') was identified which is common to the TATA-independent mRNA start sites of the human, mouse and rat tPA genes, and which demonstrates only partial similarity to sequences found at the initiation point of other TATA-independent genes.

Amino Acid Sequence↗
PubMed1989

Multiple transcription factor binding sites mediate adenovirus E1A transactivation.

We studied the response of simple synthetic promoter regions to transactivation by the adenovirus early region 1A (E1A) protein. Binding sites for one or two host cell transcription factors were substituted for the E1B promoter region in reconstructed virus mutants, and the response to E1A transactivation was assayed during the early phase of infection. We found that a single CREB/ATF binding site resulted in a surprisingly strong promoter which responded to E1A. A CREB/ATF binding site placed upstream of the E1B TATA box behaved much like the wild-type E1B promoter, which is composed of a single Sp1 binding site plus a TATA box. A single E2F binding site resulted in an extremely weak promoter which did not respond to E1A, much like a single Sp1 site. Two E2F sites in an inverted orientation with the same spacing as in the adenovirus type 2 E2 early promoter produced a strong, E1A-responsive promoter. Substitution of the E4 TATA box region for the E1B TATA box region produced a promoter about five times stronger than the wild-type E1B promoter in the absence of E1A. However, the E4 TATA box substitution did not respond significantly to E1A transactivation. These results directly demonstrate that many different transcription factor binding sites, including the E1B TATA box, a CREB/ATF binding site, and two E2F sites, can mediate E1A transactivation. Other transcription factor binding sites cannot mediate an E1A response; these other sites include the E4 TATA box, a single Sp1 binding site, and a single E2F binding site. Implications of these findings for the mechanism of E1A transactivation are discussed.

Adenovirus Early Proteins↗
PubMed1997

The downstream core promoter element, DPE, is conserved from Drosophila to humans and is recognized by TAFII60 of Drosophila.

We analyzed the function of the downstream promoter element (DPE), a distinct 7-nucleotide core promoter element that is approximately 30 nucleotides downstream of the transcription start site of many TATA-box-deficient (TATA-less) promoters in Drosophila. There is a strict requirement for spacing between the Inr and DPE motifs, as an increase or decrease of 3 nucleotides in the distance between the Inr and DPE causes a seven- to eightfold reduction in transcription as well as a significant reduction in the binding of purified TFIID. These results suggest a specific and somewhat rigid interaction of TFIID with the Inr and DPE sequences. Photo-cross-linking analysis of purified TFIID with a TATA-less DPE-containing promoter revealed specific cross-linking of dTAFII60 and dTAFII40 to the DPE, with a higher efficiency of cross-linking to dTAFII60 than to dTAFII40. These data, combined with the previously well-characterized interactions between the two TAFs and their homology to histones H4 and H3, suggest that a dTAFII60-dTAFII40 heterotetramer binds to the DPE. Human and Drosophila transcription factors exhibit essentially the same requirements for DPE sequence and for Inr-DPE spacing. In addition, the TATA-less promoter of the human interferon regulatory factor-1 (IRF-1) gene contains a DPE that is important for transcriptional activity both in vitro and in cultured cells. Hence, these studies provide evidence for a direct role of TAFs in basal transcription of TATA-less DPE-containing genes and collectively indicate that the DPE is, in many respects, a downstream counterpart to the TATA box that is present in Drosophila to humans.

Animals↗
PubMed2004

Deformations of promoter DNA bound to carcinogens help interpret effects on TATA-element structure and activity.

The TATA-box binding protein (TBP) is required by eukaryotic RNA polymerases for correct transcription initiation. TBP binds to the minor groove of an 8 base pair (bp) DNA-promoter element known as the TATA box and severely bends the TATA box. The promoter-DNA substrate can be damaged by components present in the cell or the environment to produce covalent carcinogen-DNA adducts. These may lead to transcription blockage or unfaithful transcription. Benzo[a]pyrene (BP) is a widespread environmental chemical carcinogen which can be metabolically converted to DNA-reactive enantiomeric (+) and (-)-anti-benzo[a]pyrene diol epoxides (BPDEs). Recent experimental studies of a pair of stereoisomeric adenine adducts, derived from (+) and (-)-anti-BPDEs, have revealed how these lesions influence the complexation of TBP with the TATA box. Depending on the adduct's location in the TATA box and its stereochemistry, the stability of monomeric TATA-TBP complexes was found to increase or decrease relative to the unmodified DNA. We report here analyses of molecular-dynamics simulations to interpret these findings. Structural analyses of 12 DNA-protein systems representing different combinations of adduct stereoisomer type and placement within the promoter reveal that the location of the adduct within the TATA octamer determines whether the stability of TATA-TBP complexes is increased or decreased. The effect on binding stability can be interpreted in terms of conformational freedom and major-groove space available to BP due to the hydrogen bonds and inserted phenylalanines of the TATA-TBP complex; that is, depending on the position of the adenine to which BP is covalently bound, BP can be accommodated in an intercalated or major-groove orientation with ease or with difficulty (due to interference with TATA-TBP interactions). The unravelled structures and interactions thus reveal the effect of different adduct locations on TATA-TBP complex formation and suggest how transcription initiation may be affected by the presence of a bulky BP.

Base Sequence↗
PubMed1999

Cloning and functional characterization of the 5'-flanking region of the human monocyte chemoattractant protein-1 receptor (CCR2) gene. Essential role of 5'-untranslated region in tissue-specific expression.

The human monocyte chemoattractant protein-1 receptor designated hCCR2 is an essential co-receptor in cell entry by the human immunodeficiency virus as well as a receptor for monocyte chemoattractant protein-1, a member of the family of C-C chemokines that mediate monocyte chemotaxis. To elucidate the molecular mechanisms underlying the transcriptional regulation of hCCR2, we cloned and sequenced the hCCR2 gene; it was approximately 8 kilobase pairs in length and consisted of three exons divided by two introns. In the 5'-flanking region, there were the typical mammalian promoter consensus elements, a CAAT box and a TATA box, resulting in a single transcription initiation site. In addition, we found clustered tissue-specific cis-regulatory elements such as GATA consensus sequences, Oct-1 binding sequences, and CAAT/enhancer-binding protein binding sequences. Luciferase assays with various promoter deletions and gel mobility shift assays indicated that three cis-regulatory elements located within the region from -89 to +118 are required for basal activity in THP-1 cells. One element is an octamer sequence 36-base pair upstream from the TATA box; it binds mainly to Oct-1 and is capable of increasing transcriptional activity. The other two elements, which are tandem recognition sites of the CAAT/enhancer-binding protein family, are located in the 5'-untranslated region and account for the transcriptional activation as well as the tissue specificity of hCCR2.

5' Untranslated Regions↗
PubMed1993

Genomic sequences of murine gamma B- and gamma C-crystallin-encoding genes: promoter analysis and complete evolutionary pattern of mouse, rat and human gamma-crystallins.

The murine genes, gamma B-cry and gamma C-cry, encoding the gamma B- and gamma C-crystallins, were isolated from a genomic DNA library. The complete nucleotide (nt) sequences of both genes were determined from 661 and 711 bp, respectively, upstream from the first exon to the corresponding polyadenylation sites, comprising more than 2650 and 2890 bp, respectively. The new sequences were compared to the partial cDNA sequences available for the murine gamma B-cry and gamma C-cry, as well as to the corresponding genomic sequences from rat and man, at both the nt and predicted amino acid (aa) sequence levels. In the gamma B-cry promoter region, a canonical CCAAT-box, a TATA-box, putative NF-I and C/EBP sites were detected. An R-repeat is inserted 366 bp upstream from the transcription start point. In contrast, the gamma C-cry promoter does not contain a CCAAT-box, but some other putative binding sites for transcription factors (AP-2, UBP-1, LBP-1) were located by computer analysis. The promoter regions of all six gamma-cry from mouse, rat and human, except human psi gamma F-cry, were analyzed for common sequence elements. A complex sequence element of about 70-80 bp was found in the proximal promoter, which contains a gamma-cry-specific and almost invariant sequence (crygpel) of 14 nt, and ends with the also invariant TATA-box. Within the complex sequence element, a minimum of three further features specific for the gamma A-, gamma B- and gamma D/E/F-cry genes can be defined, at least two of which were recently shown to be functional. In addition to these four sequence elements, a subtype-specific structure of inverted repeats with different-sized spacers can be deduced from the multiple sequence alignment. A phylogenetic analysis based on the promoter region, as well as the complete exon 3 of all gamma-cry from mouse, rat and man, suggests separation of only five gamma-cry subtypes (gamma A-, gamma B-, gamma C-, gamma D- and gamma E/F-cry) prior to species separation.

Amino Acid Sequence↗
PubMed2003

Archaeal chromatin and transcription.

Archaea contain a variety of sequence-independent DNA binding proteins consistent with the evolution of several different, sometimes overlapping and exchangeable solutions to the problem of genome compaction. Some of these proteins undergo residue-specific post-translational lysine acetylation or methylation, hinting at analogues of the histone modifications that regulate eukaryotic chromatin structure and transcription. Archaeal transcription initiation most closely resembles the eukaryotic RNA polymerase II (RNAPII) system, but Archaea do not appear to have homologues of the multisubunit complexes that remodel eukaryotic chromatin and activate RNAPII initiation. In contrast, they have sequence-specific regulators that repress and perhaps activate archaeal transcription by mechanisms superficially similar to the bacterial paradigm of regulating promoter binding by RNAP. Repressors compete with archaeal TATA-box binding protein (TBP) and TFB for the TATA-box and TFB-recognition elements (BRE) of the archaeal promoter, or with archaeal RNAP for the site of transcription initiation. Transcript-specific regulation by repressors binding to sites of transcript initiation is consistent with such sites having very little sequence conservation. However, most Archaea have only one TBP and/or TFB that presumably must therefore bind to similar TATA-box and BRE sequences upstream of most genes. Repressors that function by competing with TBP and/or TFB binding must therefore also make additional contacts with transcript-specific regulatory sites adjacent or remote from the TATA-box/BRE region. The fate of the archaeal TBP and TFB following transcription initiation remains to be determined. Based on functional homology with their eukaryotic RNAPII-system counterparts, archaeal TBP and possibly also TFB should remain bound to the TATA-box/BRE region after transcription initiation. However, this seems unlikely as it might limit repressor competition at this site to only the first round of transcription initiation.

Amino Acid Sequence↗
PubMed1989

Characterization of two genes for the human Na,K-ATPase beta subunit.

A total of 29 human genomic DNA clones that hybridize with cDNAs for the sheep and rat Na,K-ATPase beta subunits have been isolated, classified by restriction endonuclease mapping and Southern blot hybridization analysis, and sequenced. One class of clones, designated ATP1BL1, represents a processed pseudogene for the beta subunit. The second class, designated ATP1B, includes 15 overlapping genomic clones and represents a functional gene for the human Na,K-ATPase beta subunit. ATP1B spans about 26.7 kb of genomic DNA and includes 24 kb of intron sequence. The complete mRNA transcript for the human beta subunit is encoded by six exons, ranging in size from 81 to 1427 bp. Primer extension and S1 nuclease protection experiments with human kidney RNA indicate the presence of two major transcription initiation sites at -510 and -201 to -191, with minor initiation sites at -268, -182 to -174, and -142. The distal initiation site at -510 is preceded by consensus sequences for CAAT and TATA boxes. The DNA sequence preceding the proximal heterogeneous initiation sites contains a CAAT box, but no TATA box. Two of the 12 GC boxes (GGCGGG and CCCGCC) located in the 5' region of ATP1B are located between this CAAT box and the proximal clusters of transcription initiation sites.

Amino Acid Sequence↗
PubMed1989

Stereospecific relationships between elements in an SV40/adenovirus-2 heterologous promoter.

Spacing mutations have been constructed between an SV40 72 bp enhancer element and the Adenovirus-2 major late promoter upstream element (Ad2MLP-UE), the Ad2MLP-UE and the Ad2MLP TATA-box and the SV40 72 bp enhancer and the TATA-box in the absence of the Ad2MLP-UE. A clear periodic pattern of transcription from the Ad2MLP was obtained by inserting multiples of half of a DNA turn between the SV40 enhancer and the Ad2MLP-UE. We interpret this as a stereospecific requirement for the spatial alignment between the enhancer element and either the Ad2MLP-UE or the TATA-box. Insertions between the upstream element and the TATA-box did not give a periodic transcription pattern, though insertion of half of a DNA turn increased the steady-state level of specific RNA. Unexpectedly, spacing mutations between the enhancer and TATA-box in which the upstream element had been point mutated gave indications of stereospecificity, whereas the same insertions with a deleted upstream element showed little requirement for a precise stereoalignment.

Adenoviridae↗
PubMed2000

Transcription factor Sp1 is involved in the regulation of varicella-zoster virus glycoprotein E.

Varicella-zoster virus glycoprotein E (ORF 68) belongs to the group of late genes. It is a major component of the virion envelope and can be found complexed with glycoprotein I on the infected host cell surface. Glycoprotein E (gE) expression is activated by IE4 and IE62. Also, cellular transcription factors, like Sp1, are able to influence the gE expression. Performing quantitative reverse transcription-polymerase chain reaction, we found no decrease in Sp1 mRNA levels at different times post-infection, indicating that Sp1 mRNA evade virion host shutoff effects. In addition, the Sp1 protein was detectable in highly infected cells. Electrophoretic mobility shift assays have shown a binding of Sp1 to both GC elements within the gE-5'untranslated region (5'UTR). Additional shift assays have notified a binding of TATA box binding protein to the TATA box of the gE promoter, which is characterized by an untypical TATACA motif. Promoter-reporter constructs have been made using mutated variants of the gE-5'UTR as promoters. In transfection studies, we found that the TATA deletion, as well as inactivations of both GC boxes, reduced the basal activity of the promoter. A complete loss of activity did not become measurable until eliminating both GC elements and the TATA box, indicating that these cis-elements substitute for each other in initiation of transcription of the gE-5'UTR.

5' Untranslated Regions↗
PubMed1995

Characterization of the promoter region of a cell-adhesion protein gene derived from the basidiomycete Lentinus edodes.

An analysis of the 2 kb nucleotide sequence including the 5'-flanking region of a cell-adhesion protein-encoding gene (mfbA) isolated from the basidiomycete Lentinus edodes revealed that the promoter region contains a TATA box, a GC box, a CAAT box, a CT-rich sequence element, a TATA box, two CT-rich sequences, and a CAAT box, in the order, from upstream to downstream. Three major and three alternative transcriptional initiation sites were located 127, 129 and 131 nucleotides and 96, 193 and 197 nucleotides downstream from the downstream TATA box, and all the three major sites are positioned just in the most downstream CT-rich sequence. Three 16 bp unique sequences similar to the binding sites of Neurospora crassa transcriptional activator protein qa-1F (Baum et al. (1987) Expression of qa-1F activator protein: Identification of upstream binding sites in the qa gene cluster and localization of the DNA-binding domain. Mol. Cell. Biol. 7, 1256-1266) were present between the upstream TATA box and upstream CAAT box.

Amino Acid Sequence↗
PubMed1996

Repression of the alpha0 gene by ICP4 during a productive herpes simplex virus infection.

During a productive infection by herpes simplex virus type 1 (HSV-1), ICP4, the major regulatory protein encoded by the alpha4 gene, binds to its transcription initiation site and represses the accumulation of alpha4 RNA. Evidence suggests that the degree of repression by ICP4 is a function of the absolute distance of an ICP4 binding site 3' from a TATA box. However, repression of HSV-1 gene expression by ICP4 through binding sites located 5' of TATA boxes, as in the case of the alpha0 gene, has not been adequately addressed. To this end, recombinant alpha0 promoters with various arrays of ICP4 binding sites flanking the alpha0 TATA box were constructed and recombined into the HSV-1 genome. Our results demonstrate the following. (i) Destruction of the endogenous alphaO ICP4 binding site, located 5' of the TATA box, results in derepression of alpha0 protein and RNA accumulation in infected Vero cells. (ii) The degree of alpha0 derepression is equivalent to that reported for the alpha4 gene following destruction of the ICP4 binding site at the alpha4 mRNA cap site in HSV-1. (iii) Introduction of an ICP4 binding site at the alpha0 mRNA cap site represses the accumulation of alpha0 RNA greater than threefold relative to the wild type. (iv) Changes in the abundance of alpha0 protein and RNA in infected cells do not affect replication or growth of HSV-1 in tissue culture. Our findings are consistent with the conclusion that alpha0 transcription is repressed by ICP4. These results demonstrate that repression by ICP4 can occur through binding sites located 5' of virus gene TATA boxes in HSV-1. Thus, models addressing repression of HSV-1 gene expression by ICP4 should incorporate the role of binding sites located 5', as well as 3', of virus gene TATA boxes.

Animals↗
PubMed2001

Two-domain hemoglobin gene of the water flea Moina macrocopa: duplication in the ancestral Cladocera, diversification, and loss of a bridge intron.

Two cDNAs encoding the two-domain hemoglobin (Hb) chains of a crustacean Cladocera, Moina macrocopa, were cloned and their nucleotide (nt) sequences were determined. The amino acid (aa) sequences of both the gene products deduced from the nt sequences consisted of 348 residues and showed 98% identity with each other. These sequences together with the NH(2)-terminal aa sequences of the Hb chains determined after separation by two-dimensional gel electrophoresis showed that the Hb chains are synthesized as a secretory precursor with a signal peptide of 17 aa residues. The aa sequences of M. macrocopa Hb chains shared the following features with those of Daphnia Hb chains. Firstly, the signal peptide is followed by an NH(2)-terminal extension containing a threonine-rich sequence that might play a role in the multimerization of subunit chains. Secondly, the identity between the aa sequences of the first and second domains is exceptionally low. These facts suggest that duplication of the cladoceran Hb gene occurred before the divergence of families Moinidae and Daphniidae. Analysis of genomic DNA showed that the M. macrocopa Hb genes consist of two large repeated regions, encoding the first and second domains of Hb chains, respectively. The intron-exon organization of the first region of the M. macrocopa Hb genes was similar to that found in the Daphnia Hb genes, having the three-exon, two-intron structure characteristic of animal Hb genes. However, the intron bridging the two regions and the most downstream intron in the second region were missing in the Moina genes, providing a new example of intron loss. The following elements in the 5' flanking region were conserved in the Moina and Daphnia genes: (1) TATAAA, a typical TATA box sequence accompanied by a downstream sequence, GAAXAGCATCAGTT (the fourth residue X was G or A in Daphnia and absent in Moina); (2) CCAAT boxes, located upstream of the TATA box; (3) the binding sites for HIF-1 and GATA-1, also located upstream of the TATA box, that may be responsible for up-regulation of the cladoceran Hb genes under hypoxia.

Amino Acid Sequence↗
PubMed1994

TATA-dependent enhancer stimulation of promoter activity in mice is developmentally acquired.

Herpes simplex virus (HSV) thymidine kinase (tk) promoter activity depends on four transcription factor binding sites, one of which is a TATA box sequence, and the presence of either a cis-acting enhancer sequence or a transactivator protein. Studies presented here show that this TATA box was required for promoter activity only after cells began to differentiate and then only when promoter activity was stimulated by either an enhancer or a transactivator. When the HSV tk promoter was utilized by mouse embryos from the one-cell to eight-cell stage of development or by undifferentiated mouse embryonic stem cells, disruption of the HSV tk TATA box by site-specific mutations did not reduce promoter activity. This was true even when HSV tk promoter activity was stimulated strongly by either the embryo-responsive polyomavirus F101 enhancer or its natural transactivator, the HSV ICP4 gene product. However, stimulated expression was dependent on a distal Sp1 DNA binding site. Similarly, disruption of the TATA box did not reduce tk promoter activity in primary mouse embryonic fibroblasts or in immortalized 3T3 mouse fibroblasts; in fact, promoter activity was increased up to 2.6-fold. However, in these differentiated cells, stimulation of the HSV tk promoter by either the F101 enhancer or ICP4 protein required the TATA box. HSV tk promoter activity also was dependent on its TATA box in the mouse oocyte, a terminally differentiated cell with an endogenous transactivating activity. These results reveal that the need for a TATA box is developmentally acquired and depends on at least two parameters: the differentiated state of the cell and stimulation of the promoter by either an enhancer or a transactivator.

3T3 Cells↗
PubMed1995

The ability to associate with activation domains in vitro is not required for the TATA box-binding protein to support activated transcription in vivo.

The TATA box-binding protein (TBP) interacts in vitro with the activation domains of many viral and cellular transcription factors and has been proposed to be a direct target for transcriptional activators. We have examined the functional relevance of activator-TBP association in vitro to transcriptional activation in vivo. We show that alanine substitution mutations in a single loop of TBP can disrupt its association in vitro with the activation domains of the herpes simplex virus activator VP16 and of the human tumor suppressor protein p53; these mutations do not, however, disrupt the transcriptional response of TBP to either activation domain in vivo. Moreover, we show that a region of VP16 distinct from its activation domain can also tightly associate with TBP in vitro, but fails to activate transcription in vivo. These data suggest that the ability of TBP to interact with activation domains in vitro is not directly relevant to its ability to support activated transcription in vivo.

DNA-Binding Proteins↗
PubMed2001

High affinity interaction of yeast transcriptional regulator, Mot1, with TATA box-binding protein (TBP).

Yeast Mot1, an essential ATP-dependent regulator of basal transcription, removes TATA box-binding protein (TBP) from TATA sites in vitro. Complexes of Mot1 and Spt15 (yeast TBP), radiolabeled in vitro, were immunoprecipitated with anti-TBP (or anti-Mot1) antibodies in the absence of DNA, showing Mot1 binds TBP in solution. Mot1 N-terminal deletions (residues 25-801) abolished TBP binding, whereas C-terminal ATPase domain deletions (residues 802-1867) did not. Complex formation was prevented above 200 mm salt, consistent with electrostatic interaction. Correspondingly, TBP variants lacking solvent-exposed positive charge did not bind Mot1, whereas a mutant lacking positive charge within the DNA-binding groove bound Mot1. ATPase-defective mutant, Mot1(D1408N), which inhibits growth when overexpressed (but is suppressed by co-overexpression of TBP), bound TBP normally in vitro, suggesting it forms nonrecyclable complexes. N-terminal deletions of Mot1(D1408N) were not growth-inhibitory. C-terminal deletions were toxic when overexpressed, and toxicity was ameliorated by TBP co-overproduction. Residues 1-800 of Mot1 are therefore necessary and sufficient for TBP binding. The N terminus of 89B, a tissue-specific Drosophila Mot1 homolog, bound the TBP-like factor, dTRF1. Native Mot1 and derivatives deleterious to growth localized in the nucleus, whereas nontoxic derivatives localized to the cytosol, suggesting TBP binding and nuclear transport of Mot1 are coupled.

Adenosine Triphosphatases↗
PubMed1998

Promoter analysis of the Drosophila genes encoding TFIIB and TATA box-binding protein.

We have analyzed the 5'-flanking regions of the Drosophila genes encoding the TFIIB and TATA box-binding proteins (TBP) and mapped the regulatory regions required for their efficient expression. We found that the 500 bp long region (-439 to +60) and the 401 bp long region (-261 to +138) are required for the efficient expression of TFIIB and TBP genes, respectively. In the TFIIB promoter region, the upstream DNA between -439 and -280 and the downstream DNA between +8 and +60 are necessary for the stimulation of promoter activity. The upstream DNA between -439 and -280 stimulates transcription in an orientation dependent manner. In the TBP promoter region, the upstream DNA between -261 and -207, and the downstream DNA between +15 and +138 are necessary for the stimulation of promoter activity. The upstream DNA (-261 to -207) required for TBP promoter activity contains a 11 bp long palindromic sequence and a DNA replication-related element sequence. Particularly, we could find that the downstream promoter regions of TFIIB and TBP genes contain the conserved nucleotide sequences, suggesting the presence of a common regulatory mechanism for the expression of these two genes.

Animals↗
PubMed2000

An initiation element in the yeast CUP1 promoter is recognized by RNA polymerase II in the absence of TATA box-binding protein if the DNA is negatively supercoiled.

Purified RNA polymerase II initiated transcription from the yeast CUP1 promoter fused to a C-less cassette if the DNA was negatively supercoiled. Relaxed plasmid was not transcribed. Transcription did not require addition of any other transcription factors. TATA box-binding protein (TBP) was not detectable in the polymerase preparation and the TATA box was not required. Deletion analysis of the CUP1 promoter revealed that a 25-bp element containing the initiation region was sufficient for recognition by polymerase. Two transcription start sites were mapped, one of which is identical to one of the two major start sites observed in vivo. Our observations can be accounted for by using a theoretical analysis of the probability of DNA melting within the plasmid as a function of superhelix density: the CUP1 initiation element is intrinsically unstable to superhelical stress, permitting entry of the polymerase, which then scans the DNA to locate the start site. In support of this analysis, the CUP1 promoter was sensitive to mung bean nuclease. These observations and a previous theoretical analysis of yeast genes support the idea that promoters are stress points within the DNA superhelix. The role of transcription factors might be to mark the promoter and to regulate specific melting of promoter DNA.

Carrier Proteins↗