SEARCH · PubMed Health

Results for “Promotion”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 37 records · Page 2Linked to original sources

PubMed1991

Regulation of the multiple promoters of the human aldolase A gene: response of its two ubiquitous promoters to agents promoting cell proliferation.

The human aldolase A gene is transcribed from three distinct promoters, the two ubiquitous promoters PN and PH and the muscle specific promoter PM. In the present study, we investigate further aldolase A mRNA structure and expression. We demonstrate that the upstream N-type exon is, in fact, extremely heterogeneous. RNAse H mapping experiments permit quantification of relative abundance of N, M, and H type mRNAs and show that the level of transcripts containing the downstream H-type exon is at least 30 times higher than that of those containing N exon, in all tissues tested. Aldolase A level is up-regulated in proliferating cells. Here we show that both N and H type mRNAs, although barely detectable in normal liver, are highly expressed in human hepatomas biopsies. Furthermore, in human lymphocytes, N-type mRNA level is enhanced by serum treatment, while in cultured Hep G2 cells, both N-type and H-type mRNA levels are increased by serum and by the tumor promoting agent PMA. Using CAT constructs in transfection experiments, we demonstrate that the H exon plus its upstream region can function autonomously: the 420 base pairs upstream of the H exon are sufficient to confer to promoter PH an efficiency comparable that of the complete SV40 early promoter and enhancer in two cell lines.

Base Sequence↗

PubMed1996

Upstream interactions at the lambda pRM promoter are sequence nonspecific and activate the promoter to a lesser extent than an introduced UP element of an rRNA promoter.

The rightward regulatory region of bacteriophage lambda contains two promoters, pRM and pR, which direct the synthesis of nonoverlapping divergent transcripts from start sites 82 bp apart. Each of the two promoters has an upstream (A+T)-rich region (ATR) within the sequence from -40 to -60 where in the rrnB P1 promoter a stretch of 20 (A+T) bp greatly stimulates promoter function. Here we present an investigation of the possible functional significance of pRM's ATR. We determined the effects on RNA polymerase-pRM promoter interaction both of (G+C) substitutions in the ATR and of amino acid substitutions in the alpha subunit, known to affect the upstream interaction. We find small (two- to threefold) effects of selected mutations in the alpha subunit on open complex formation at pRM. However, the (presumably upstream) interactions underlying these effects are sequence nonspecific, as they are not affected by (G+C) substitutions in the ATR. Substitution of the 20-bp UP element of the rrnB P1 promoter between positions -40 and -60 at pRM stimulates open complex formation to a considerably greater extent (5- to 10-fold). Results from kinetic studies indicate that on this construct the UP element mainly accelerates a step subsequent to the binding of RNA polymerase, although it may also facilitate the binding event itself. Less extensive studies likewise provide evidence for a two- to threefold activation of pR by upstream interactions. The possible involvement of the alpha subunit in the previously characterized (e.g., B. C. Mita, Y. Tang, and P. L. deHaseth, J. Biol. Chem. 270:30428-30433, 1995) interference of pR-bound RNA polymerase with open complex formation at pRM is discussed.

Amino Acid Sequence↗

PubMed1999

The human prolactin receptor gene structure and alternative promoter utilization: the generic promoter hPIII and a novel human promoter hP(N).

The 5'-untranslated region of the human prolactin receptor (hPRLR) gene contains two alternative first exons, hE1(3), the human counterpart of the rat and mouse E1(3) and a novel human type of alternative first exon termed hE1N, also a common non-coding exon 2 and a third exon containing the translation initiation codon. hE1(3) was localized approximately 800 bp 5' from the hE1N in the genome. The two distinct first exons hE1(3) and hE1N are expressed in human breast tissue, breast cancer cells, gonads and liver. Overall, the transcript containing hE1(3) is prevalent in most tissues. The coding region of the gene comprises eight exons (exon 3-10), in which exon 10 encodes most of the intracellular domain. hE1(3) and hE1N are transcribed from alternative promoters hPIII and hP(N), respectively. The hPIII, containing identical Sp1 and C/EBP elements as in the rodent promoters, shares 81% similarity in the region -480/-106 to both the rat and mouse. The novel promoter hP(N) contains putative binding sites for ETS-family proteins and a half-site for nuclear receptors. Therefore, both promoters likely utilize distinct mechanisms in controlling the hPRLR gene transcription. The different promoter utilization of the hPRLR gene in diverse tissues may confer differential prolactin response through activation of different promoters.

Base Sequence↗

PubMed1995

Evaluation in tobacco of the organ specificity and strength of the rolD promoter, domain A of the 35S promoter and the 35S2 promoter.

In order to study the expression in plants of the rolD promoter of Agrobacterium rhizogenes, we have constructed chimaeric genes placing the coding region of the gusA (uidA) marker gene under control of two rolD promoter fragments of different length. Similar results were obtained with both genes. Expression studies were carried out in transformed R1 progeny plants. In mature transformed tobacco plants, the rolD-gus genes were expressed strongly in roots, and to much lower levels in stems and leaves. This pattern of expression was transmitted to progeny, though the ratio of the level of expression in roots relative to that in leaves was much lower in young seedlings. The degree of root specificity in rolD-gus transformants was less than that of a gene constructed with domain A of the CaMV 35S promoter, domA-gus, but the level of root expression was much higher than with the latter gene. However, the level of expression of the rolD-gus genes was less than that of a gus gene with a 35S promoter with doubled domain B, 35S2-gus. The rolD-gus genes had a distinctive pattern of expression in roots, compared to that of the two other genes, with the strongest GUS activity observed in the root elongation zone and in vascular tissue, and much less in the root apex.

Base Sequence↗

PubMed2006

The rcsA promoter of Pantoea stewartii subsp. stewartii features a low-level constitutive promoter and an EsaR quorum-sensing-regulated promoter.

The upstream region of the Pantoea stewartii rcsA gene features two promoters, one for constitutive basal-level expression and a second autoregulated promoter for induced expression. The EsaR quorum-sensing repressor binds to a site centered between the two promoters, blocking transcription elongation from the regulated promoter under noninducing conditions.

Bacterial Proteins↗

PubMed2003

Chemoprevention of DMBA-induced UV-B promoted, NOR-1-induced TPA promoted skin carcinogenesis, and DEN-induced phenobarbital promoted liver tumors in mice by extract of beetroot.

Our previous studies identified the extract of Beta vulgaris (beetroot), commercially also known as betanin, as a potent cancer chemopreventive agent in both in vitro Epstein-Barr early antigen activation assay and in an in vivo two-stage mouse lung and skin carcinogenesis. To explore this issue further, we have now investigated its cancer chemopreventive potentials in three different chemical carcinogen initiation-promotion experimental tumor models in mice. Following tumor initiation with 390 nmol of 7,12-dimethylbenz(a)anthracene (DMBA) in 100 microl of acetone, the mouse skin tumor promotion with 3430 J/m(2) of ultraviolet light-B (UV-B) as well as splenomegaly was significantly inhibited by oral administration of 0.0025% betanin. At the same dose, betanin also afforded significant protection in the mouse skin cancer model following the topical application of 390 nmol of (+/-)-(E)-4-methyl-2-[(E)-hydroxyamino]-5-nitro-6-methoxy-3-hexanamide (NOR-1) in 100 microl of acetone and promoted by topical administration of 1.7 nmol of 12-O-tetradecanoylphorbol-13-acetate (TPA). In the two-stage model of hepatocarcinogenesis in mice with N-nitrosodiethylamine (DEN, 30 mg/kg) as the initiator and phenobarbital as the promoter, oral administration of 0.0025% betanin also showed a very significant inhibition of both the incidence and multiplicity of the liver tumors. These findings along with our initial reports suggest that betanin which is a regularly consumed natural product colorant is an effective cancer chemopreventive agent in mice. The most interesting observation is that the cancer chemopreventive effect was exhibited at a very low dose used in the study and thus indicating that beetroot warrants more attention for possible human applications in the control of malignancy.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1989

Eicosanoids and multistage carcinogenesis in NMRI mouse skin: role of prostaglandins E and F in conversion (first stage of tumor promotion) and promotion (second stage of tumor promotion).

When applied to NMRI mouse skin in vivo, phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and 12-O-retinoylphorbol-13-acetate (RPA) have been found to induce two early waves of prostaglandin E2 (PGE2) synthesis after 15 and 90 min and a delayed accumulation of prostaglandin F2 alpha (PGF2 alpha) after 2 h. With respect to PGF2 alpha formation different activities of both agents were observed, in that TPA but not RPA induced additional PGF2 alpha waves after 4 and 17 h. Functionally, PGE2 was previously shown to be an endogenous mediator of the TPA- or RPA-induced epidermal hyperproliferation and hyperplasia. A functional role of PGF2 alpha could be attributed to the post-initiation stages of tumor development in initiated mouse skin, i.e. the conversion stage (stage I of tumor promotion) elicited by two TPA applications and the promotion stage (stage II of promotion) brought about repetitive RPA treatments. PGF2 alpha, appearing as one distinct biosynthetic wave 3-4 h after TPA application seems to be critically involved in the conversion steps since (i) inhibition of its accumulation by indomethacin led to an inhibition of tumor formation, (ii) the inhibitory effect of indomethacin could be reversed by PGF2 alpha and (iii) RPA was not able to give rise to the accumulation of PGF2 alpha 4 h after application as obtained by TPA treatment. Moreover, RPA-induced promotion of DMBA- and TPA-treated mouse skin was inhibited by indomethacin. The inhibitory effect of indomethacin on papilloma formation was again reversed by PGF2 alpha treatment concomitant with RPA.

Animals↗

PubMed1991

Analysis of Sindbis virus promoter recognition in vivo, using novel vectors with two subgenomic mRNA promoters.

Four types of Sindbis virus vectors, each carrying two promoters for subgenomic mRNA synthesis, were designed to measure relative promoter strengths and to survey potential contextual effects on promoter strengths. One of the promoters in each vector was used as the reference promoter, while the other was the one being tested. We used these vectors to measure the relative strengths of four promoters: the minimal promoter, an extended sequence believed to have full promoter activity, and two mutant promoters, one with an inactivating 3-nucleotide insertion called CR4.1 and the other with a 4-nucleotide deletion called delta 4. The strengths of the promoters were measured by quantitating the RNA transcribed from each promoter in vivo and also by assaying for chloramphenicol acetyltransferase activity encoded by one of the two transcripts. We found that the relative strengths of the promoters were similar in different contexts. The complete promoter was 6-fold more active, the delta 4 promoter was (surprisingly) about twice as active, and the CR4.1 promoter was 100-fold less active than the minimal promoter. At least two contextual effects were identified that can alter the activity of one or both promoters in the vectors. One effect is that given identical promoters, the 3'-proximal promoter on the minus-strand template can be more active than the 5'-proximal promoter. This may be due to preferential association of one or more components of the transcription complex for the 3' end of the minus-strand template. A second effect is promoter competition, particularly when the promoters are closely spaced.

Animals↗

PubMed1998

A CACCC box in the proximal exon 2 promoter of the rat insulin-like growth factor I gene is required for basal promoter activity.

The insulin-like growth factor I gene is transcribed from two promoter regions, resulting in alternative first exons in insulin-like growth factor I messenger RNAs. A previous study showed that the sequence from -73 to +44 (where +1 is the first nucleotide in the exon 2 transcription initiation cluster) contained an active exon 2 promoter, and that sequences between -73 and -36 were required for promoter activity. In the current study, the roles of two putative cis-acting elements within the -73 to +44 region in basal exon 2 promoter activity were evaluated using mutagenesis and nuclear protein-DNA binding assays. Mutation of the CCCCACCC sequence at position -53 to GAAATCCC resulted in a complete loss of promoter activity in transient transfection assays in GH3, OVCAR-3, C6, and Chinese hamster ovary (CHO) cells. A -73/+24 exon 2 promoter-luciferase construct had partial promoter activity. Mutation of a putative initiator motif surrounding the major exon 2 start site did not alter the activity of this construct. In electrophoretic mobility shift assays, a 32P-labeled oligomer extending from -73 to +44 in the exon 2 promoter was specifically bound by GH3 cell nuclear extracts. A 32P-labeled oligomer which extended from -63 to -37 in the exon 2 promoter was specifically bound by GH3 and OVCAR-3 cell nuclear extracts. These unlabeled oligomers inhibited the binding of a labeled -236/+44 exon 2 promoter fragment to OVCAR-3 nuclear extracts. Mutation of the CCCCACCC sequence prevented the unlabeled -73/+44 oligomer from inhibiting the binding of the -236/+44 fragment. An unlabeled oligomer containing a consensus activating protein-2 (AP-2)-binding site inhibited labeled -236/+44, -73/+44, and -63/-37 exon 2 promoter binding with a much lower affinity than did the respective unlabeled oligomers. Purified AP-2 protein did not bind to the exon 2 promoter fragment, nor did anti-AP-2 antibody alter the binding. Cotransfection of AP-2 expression vector did not significantly increase exon 2 promoter activity. On the other hand, an oligomer containing a consensus Sp1-binding site inhibited labeled -63/-37 exon 2 promoter binding by GH3 cell nuclear extracts with an affinity similar to that of the unlabeled -63/-37 oligomer. A mutation in the Sp1-binding site in this same oligomer resulted in a complete loss of binding affinity. Purified Sp1 bound to the -63/-37 exon 2 promoter oligomer. Addition of polyclonal antibody to Sp1 resulted in a partial supershift of the complex formed between GH3 cell and OVCAR-3 cell nuclear extracts and the labeled -63/-37 oligomer. However, in Drosophila Schneider cells, which are an experimental model for studying the ability of Sp1 to activate transcription, the -73/+44 exon 2 promoter construct was not stimulated by cotransfection with an Sp1 expression plasmid. UV cross-linking studies indicated that proteins of approximate molecular mass 125, 76, 47, and 38 kDa are bound to the proximal (-236/+44) exon 2 promoter region. It is concluded that the CCCCACCC sequence at -53 is required for exon 2 promoter activity. Moreover, specific binding of nuclear proteins to the proximal exon 2 promoter region requires the CCCCACCC sequence. Sequences downstream of the exon 2 initiation site from +24 to +44 are required for full promoter activity. However, the putative initiator surrounding the major transcription start site at +1 does not appear to be important for the strength of the proximal promoter. The CCCCACCC sequence at -53 appears to be a CACCC box, which binds zinc finger transcription factors of the Kruppel family such as Sp1, although protein factors in addition to Sp1 are required to activate exon 2 transcription through the -73/+44 proximal promoter region.

Animals↗

PubMed1995

PARP promoter-mediated activation of a VSG expression site promoter in insect form Trypanosoma brucei.

In trypanosomes the rRNA, PARP and VSG gene promoters mediate alpha-amanitin-resistant transcription of protein coding genes, presumably by RNA polymerase (pol) I. We compared the activity of PARP and VSG promoters integrated at one of the alleles of the largest subunit of pol II genes in insect form trypanosomes. Even though both promoters are roughly equally active in transient transformation assays in insect form trypanosomes, only the PARP promoter functioned effectively when integrated at the pol II largest subunit or other loci. Promoter activity in transient transformation assays is therefore not necessarily predictive of transcriptional activity once integrated into the trypanosome genome. The integrated fully active PARP promoter could upregulate in cis an otherwise poorly active integrated VSG promoter. The PARP promoter nucleotide sequence elements responsible for VSG promoter activation coincided with most of the important PARP promoter elements mapped previously by linker scanning mutagenesis, indicating that it is not a single unique promoter element that was responsible for VSG promoter activation. The data suggest that PARP promoter-mediated activation of the VSG promoter does not result from complementation of the VSG promoter with a single insect form-specific transcription factor whose binding site is missing from the VSG promoter and present in the PARP promoter. We favor a model in which chromatin structure at the locus is altered by the PARP promoter, allowing VSG promoter activation in insect form trypanosomes. We discuss the significance of these observations for the control of VSG promoters in insect form trypanosomes.

Animals↗

PubMed1993

Comparison of promoter activity in Aleutian mink disease parvovirus, minute virus of mice, and canine parvovirus: possible role of weak promoters in the pathogenesis of Aleutian mink disease parvovirus infection.

Aleutian mink disease parvovirus (ADV) infection causes both acute and chronic disease in mink, and we have previously shown that it is the level of viral gene expression that determines the disease pattern. To study the gene regulation of ADV, we have cloned the P3 ADV and P36 ADV promoters in front of a reporter gene, the chloramphenicol acetyltransferase (CAT) gene, and analyzed these constructs by transient transfection in a feline kidney cell line and mouse NIH 3T3 cells. The genes for ADV structural proteins (VP1 and VP2) and the nonstructural proteins (NS-1, NS-2, and NS-3) were cloned into a eukaryotic expression vector, and their functions in regulation of the P3 ADV and P36 ADV promoters were examined in cotransfection experiments. The ADV NS-1 protein was able to transactivate the P36 ADV promoter and, to a lesser degree, the P3 ADV promoter. Constitutive activities of the P3 ADV and P36 ADV promoters were weaker than those of the corresponding promoters from the prototypic parvovirus minute virus of mice (MVM) and canine parvovirus (CPV). Also, the level of transactivation of the P36 ADV promoter was much lower than those of the corresponding P38 MVM and P38 CPV promoters transactivated with MVM NS-1. Moreover, the ADV NS-1 gene product could transactivate the P38 MVM promoter to higher levels than it could transactivate the P36 ADV promoter, while the P36 ADV promoter could be transactivated by MVM NS-1 and ADV NS-1 to similar levels. Taken together, these data indicated that cis-acting sequences in the P36 ADV promoter play a major role in determining the low level of transactivation observed. The P3 ADV and P4 MVM promoters could be transactivated to some degree by their respective NS-1 gene products. However, in contrast to the situation for the late promoters, switching NS-1 proteins between the two viruses was not possible. This finding may indicate a different mechanism of transactivation of the early promoters (P3 ADV and P4 MVM) compared with the late (P36 ADV and P38 MVM) promoters. In summary, the constitutive levels of expression from the ADV promoters are weaker than the levels from the corresponding promoters of MVM and CPV. Moreover, the level of NS-1-mediated transactivation of the late ADV promoter is impaired compared with the level of transactivation of the late promoters of MVM and CPV.(ABSTRACT TRUNCATED AT 400 WORDS)

3T3 Cells↗

PubMed1992

The efficiency of promoter clearance distinguishes T7 class II and class III promoters.

Promoter strength has been defined as the relative production of transcripts from a promoter. For T7 transcription it has frequently been observed that T7 class III promoters are qualitatively stronger than T7 class II promoters. In previous work it was observed that the maximum rates of initiation of three class III and three class II promoters show no class distinctions (Ikeda, R. A., Lin, A. C., and Clarke, J. (1992) J. Biol. Chem. 267, 2640-2649). This suggests that the efficiency of the conversion of the polymerase initiation complex to a stable transcription complex contributes to the overall strength of T7 promoters. The class differences in the strengths of T7 class II and class III promoters are confirmed by measuring the relative synthesis of run-off transcripts. These results show that the relative strengths of the class III promoters, phi 6.5, phi 10, and phi 13, are all comparable ranging from 0.61 for phi 6.5 to 1.00 for phi 10, while the relative strengths of the T7 class II promoters, phi 1.1B, phi 1.3, and phi 3.8, vary widely. One T7 class II promoter, phi 1.1B (relative strength = 0.34), approaches the strength of the class III promoters, while the other T7 class II promoters, phi 1.3 (relative strength = 0.045) and phi 3.8 (relative strength = 0.070) are nearly inactive. The efficiency of promoter clearance is then determined by measuring the relative production of small transcription products in comparison with the production of run-off transcripts. These measurements clearly distinguish the T7 class III promoters from the T7 class II promoters. It is found that 68-75% of all initiations at the T7 class III promoters phi 6.5, phi 10, and phi 13 produce a run-off transcript, while only 16-36% of the initiations at the T7 class II promoters phi 1.1B, phi 1.3, and phi 3.8 produce a run-off transcript. Clearly, promoter clearance contributes to the difference in promoter strengths of the T7 class II and III promoters.

Base Sequence↗

PubMed2004

Only a subset of 12-O-tetradecanoylphorbol-13-acetate-promoted mouse skin papillomas are promotable by benzoyl peroxide.

The two-stage skin carcinogenesis model of initiation and promotion in Carcinogenesis-susceptible (Car-S) mice has been used to investigate the pathways of promotional activity of 12-O-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester tumor promoter, and benzoyl peroxide (BzPo), a free radical-generating compound. To test whether distinct populations of 9,10-dimethyl-1,2-benzanthracene (DMBA)-initiated epidermal keratinocytes are responsive to the two promoters, tandem experiments were performed. DMBA-initiated Car-S mice were promoted twice weekly with maximal promoting doses of TPA or BzPo. When the number of papillomas/mouse reached a plateau, promotion in the TPA and BzPo groups was switched to BzPo or TPA, respectively, until achievement of a new plateau. Mice promoted with BzPo developed 11.0 +/- 1.3 papillomas/mouse and subsequent TPA promotion induced 13.8 additional papillomas, for a total of 24.8 +/- 2.1 papillomas/mouse. TPA-promoted mice developed 23.3 +/- 1.1 papillomas/mouse, and subsequent BzPo promotion for 91 days did not promote additional papillomas. Our results show a less than additive tumor response after sequential promotion with BzPo and TPA, or vice versa, indicating that the pathways of promotional activity of TPA and BzPo are interacting. While the final papilloma yield was similar at the end of the two tandem promotion experiments independently of promoter sequence, the percentage of mice developing carcinomas was significantly higher in mice that were promoted with BzPo in the first stage. No significant differences in the frequency and type of c-Ha-ras mutations were observed in TPA- and BzPo-promoted tumors, suggesting that promotion of DMBA-initiated cells by BzPo requires introduction of additional molecular alterations compared to TPA.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed2003

Characterization of an oxygen-dependent inducible promoter, the Escherichia coli nar promoter, in gram-negative host strains.

The Escherichia coli nar promoter is maximally induced under anaerobic conditions in the presence of nitrate ion or under anaerobic only conditions, depending on the genotype of the E. coli nar promoter. Previously, we found that the E. coli nar promoter has some desirable characteristics as an inducible promoter in the E. coli host strains. In this study, the E. coli nar promoter with lacZ gene at the downstream was cloned onto a broad-host-range Gram-negative vector, pBBR122. It was then induced in some other Gram-negative host strains, such as Agrobacterium, Pseudomonas, and Rhizobium, to determine whether the E. coli nar promoter could be used as an inducible promoter in these strains. From shake-flask experiments it was found that the wild-type E. coli nar promoter cloned onto pBBR122, pNW61, was suppressed under aerobic conditions in an Agrobacterium host strain, was partially induced under microaerobic only conditions, and was maximally induced under microaerobic conditions in the presence of nitrate ion. Whereas the mutant-type E. coli nar promoter cloned onto pBBR122, pNW618, was suppressed under aerobic conditions and was maximally induced under microaerobic conditions, regardless of the presence of nitrate ion. This kind of induction pattern observed for the E. coli nar promoters in the Agrobacterium host strain was similar to that observed for the E. coli nar promoters in the E. coli host strain. On the other hand, it was found that both of the E. coli nar promoters, pNW61 and pNW618, in a Pseudomonas host strain were partially induced under aerobic conditions and were maximally induced under microaerobic conditions, regardless of the presence of nitrate. Finally, it was found that both of the E. coli nar promoters in a Rhizobium host strain were minimally induced, regardless of the presence of oxygen or nitrate ion. Similar induction patterns for the three strains were also observed from fermentor experiments in which the dissolved oxygen (DO) level was tightly controlled. From an evolutionary point of view, the results from the three Gram-negative host strains indicate that the E. coli nar promoter system, including the promoter and regulatory proteins, was best conserved in the Agrobacterium host strain and the least conserved in the Rhizobium host strain. From an industrial point of view, the results indicate that the E. coli nar promoter system can be used as an oxygen-dependent inducible promoter in both Agrobacterium and Pseudomonas host strains.

Cloning, Molecular↗

PubMed2003

Sigma70 promoters in Escherichia coli: specific transcription in dense regions of overlapping promoter-like signals.

We present here a computational analysis showing that sigma70 house-keeping promoters are located within zones with high densities of promoter-like signals in Escherichia coli, and we introduce strategies that allow for the correct computer prediction of sigma70 promoters. Based on 599 experimentally verified promoters of E.coli K-12, we generated and evaluated more than 200 weight matrices optimizing different criteria to obtain the best recognition matrices. The alignments generating the best statistical models did not fully correspond with the canonical sigma70 model. However, matrices that correspond to such a canonical model performed better as tools for prediction. We tested the predictive capacity of these matrices on 250 bp long regions upstream of gene starts, where 90% of the known promoters occur. The computational matrix models generated an average of 38 promoter-like signals within each 250 bp region. In more than 50% of the cases, the true promoter does not have the best score within the region. We observed, in fact, that real promoters occur mostly within regions with high densities of overlapping putative promoters. We evaluated several strategies to identify promoters. The best one uses an intrinsic score of the -10 and -35 hexamers that form the promoter as well as an extrinsic score that uses the distribution of promoters from the start of the gene. We were able to identify 86% true promoters correctly, generating an average of 4.7 putative promoters per region as output, of which 3.7, on average, exist in clusters, as a series of overlapping potentially competing RNA polymerase-binding sites. As far as we know, this is the highest predictive capability reported so far. This high signal density is found mainly within regions upstream of genes, contrasting with coding regions and regions located between convergently transcribed genes. These results are consistent with experimental evidence that show the existence of multiple overlapping promoter sites that become functional under particular conditions. This density is probably the consequence of a rich number of vestiges of promoters in evolution. We suggest that transcriptional regulators as well as other functional promoters play an important role in keeping these latent signals suppressed.

Bacterial Proteins↗

PubMed1996

Silencing of the Escherichia coli bgl promoter: effects of template supercoiling and cell extracts on promoter activity in vitro.

Regulation of the Escherichia coli bgl promoter involves the catabolite gene activator protein (CAP) and silencer elements that are located upstream and downstream of the promoter and its CAP binding site. The promoter is kept in a repressed state by the silencer elements and other normally active CAP-dependent or -independent promoters are repressed as well when flanked by these elements. To assess the mechanism of promoter repression, single round in vitro transcription was carried out with plasmids bearing either the wild-type bgl promoter or one of two derivatives that escape repression in vivo by different mechanisms: C234 by improving the CAP binding site of the promoter and delta1 by a deletion within the upstream silencer sequence. Repression of the bgl promoter in vitro was shown to depend on template topology and the presence of cellular factors. With negatively supercoiled templates, all three promoters are transcribed to similar extents by purified E. coli RNA polymerase and no CAP dependence is apparent; with relaxed templates, transcription is CAP dependent, but the levels of transcription of the three promoters are comparable. Addition of crude cell extract to the simple transcription system leads to repression of all three promoter alleles in the absence of CAP. Repression of the mutant alleles but not of the wild-type promoter is completely relieved in the presence of the CAP-cAMP complex. The topology of the DNA template is also important in the differential regulation of these promoters. In the case of C234, repression by cell extract is completely relieved by CAP-cAMP on relaxed or negatively supercoiled templates, while complete derepression of delta1 by CAP-cAMP occurs on negatively supercoiled templates only. Repression by cell extract requires the presence of the histone-like protein H-NS. However, H-NS alone does not appear to be sufficient for specific silencing of the wild-type promoter, since repression of all three promoter alleles caused by purified H-NS protein is completely relieved by the CAP-cAMP complex. These data suggest that template topology, H-NS and other cellular factors are involved in the formation of a specific nucleoprotein structure in the bgl promoter-silencer region; the formation of this nucleoprotein structure keeps an otherwise active promoter in an inactive state.

Amino Acid Sequence↗

PubMed2003

Mutational analysis of the Chlamydia trachomatis dnaK promoter defines the optimal -35 promoter element.

A long-standing question in the biology of the intracellular bacterium, Chlamydia, has been the structure of the promoter recognized by its RNA polymerase. The 'RNA polymerase sigma subunit paradox' refers to the difficulty reconciling the conservation between the RNA polymerases of Chlamydia and Escherichia coli, especially at the level of the promoter-recognition sigma subunit, with the general lack of homology between chlamydial promoters and the E.coli sigma(70) consensus promoter. While the -10 promoter element appears to be conserved between Chlamydia and E.coli, the structure of the chlamydial -35 promoter element has not been defined. We have investigated the structure of the -35 element of the Chlamydia trachomatis dnaK promoter by measuring the effects of single base pair substitutions on in vitro promoter activity. Most substitutions produced large decreases in promoter activity, which allowed us to define the optimal -35 sequence in the context of the dnaK promoter. We found that the optimal chlamydial -35 promoter sequence is identical to the E.coli sigma(70) consensus -35 promoter element (TTGACA). These results indicate that the optimal promoter specificities of the major form of chlamydial RNA polymerase and E.coli sigma(70) RNA polymerase are in fact highly conserved. A further implication of our results is that many chlamydial promoters have a suboptimal promoter structure. We hypothesize that these chlamydial promoters are intrinsically weak promoters that can be regulated during the chlamydial developmental cycle by additional transcription factors.

Base Sequence↗

PubMed1997

Characterization of the rat insulin-like growth factor I gene promoters and identification of a minimal exon 2 promoter.

Insulin-like growth factor I (IGF-I) promoter activity was characterized in C6, GH3, OVCAR-3, and Chinese hamster ovary (CHO) cells. Maximal exon 1 promoter activity was present in the region extending from -133 to +362 (where +1 is the first transcription start site). Promoter activity was higher in the +75/+362 fragment, which contains exon 1 transcription start sites 3 and 4, than in the -133/+74 fragment, which contains exon 1 transcription start sites 1 and 2. Promoter activity was also observed in constructs containing sequences from -133 to +192, which includes start sites 1, 2, and 3. Inclusion of sequences upstream of -133 inhibited exon 1 proximal promoter activity in a cell type-specific manner. Exon 2 promoter activity was observed in all cell lines with a construct containing 73 bp of 5'-flanking sequence and 44 bp of exon 2. Exon 2 promoter activity was abolished when only 36 bp of 5'-flanking sequence and 44 bp of exon 2 were present, suggesting that an essential minimal promoter element(s) is contained within the -73 to -36 region. A putative CACCC box was observed within this region at -53. Upstream sequence regulated exon 2 promoter activity in a cell type-specific manner. Electrophoretic mobility shift assays revealed a single specifically bound band when the +75/+362 fragment of the exon 1 promoter was used with nuclear extracts from C6 and GH3 cells. Multiple specifically bound bands with slower mobility were observed when the -236/+44 exon 2 promoter fragment was incubated with C6, GH3, CHO, and OVCAR-3 cell nuclear extracts. The exon 1 and exon 2 promoter regions were able to inhibit each other's binding in electrophoretic mobility shift assay using GH3 cell and OVCAR-3 cell nuclear extracts, respectively. Oligonucleotides containing consensus activating protein-1 (AP-1) and AP-3 sequences inhibited exon 1 promoter binding by GH3 cell nuclear extracts. AP-2 and AP-3 sites inhibited exon 2 promoter binding. Our data suggest that the sequence surrounding and including start site 3 in exon 1 functions as a minimal independent promoter. The minimal exon 2 promoter is contained within the 73 bp upstream and 44 bp downstream of the transcription start site cluster. These minimal promoters contain similar and distinct elements that are important for basal transcription. Upstream sequences may contain cell type-specific silencer elements.

Animals↗