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PubMed2000

The downstream promoter element DPE appears to be as widely used as the TATA box in Drosophila core promoters.

The downstream promoter element (DPE) functions cooperatively with the initiator (Inr) for the binding of TFIID in the transcription of core promoters in the absence of a TATA box. We examined the properties of sequences that can function as a DPE as well as the range of promoters that use the DPE as a core promoter element. By using an in vitro transcription assay, we identified 17 new DPE-dependent promoters and found that all possessed identical spacing between the Inr and DPE. Moreover, mutational analysis indicated that the insertion or deletion of a single nucleotide between the Inr and DPE causes a reduction in transcriptional activity and TFIID binding. To explore the range of sequences that can function as a DPE, we constructed and analyzed randomized promoter libraries. These experiments yielded the DPE functional range set, which represents sequences that contribute to or are compatible with DPE function. We then analyzed the DPE functional range set in conjunction with a Drosophila core promoter database that we compiled from 205 promoters with accurately mapped start sites. Somewhat surprisingly, the DPE sequence motif is as common as the TATA box in Drosophila promoters. There is, in addition, a striking adherence of Inr sequences to the Inr consensus in DPE-containing promoters relative to DPE-less promoters. Furthermore, statistical and biochemical analyses indicated that a G nucleotide between the Inr and DPE contributes to transcription from DPE-containing promoters. Thus, these data reveal that the DPE exhibits a strict spacing requirement yet some sequence flexibility and appears to be as widely used as the TATA box in Drosophila.

ATP-Binding Cassette Transporters↗
PubMed2000

c-Myb binds to a sequence in the proximal region of the RAG-2 promoter and is essential for promoter activity in T-lineage cells.

The RAG-2 gene encodes a component of the V(D)J recombinase which is essential for the assembly of antigen receptor genes in B and T lymphocytes. Previously, we reported that the transcription factor BSAP (PAX-5) regulates the murine RAG-2 promoter in B-cell lines. A partially overlapping but distinct region of the proximal RAG-2 promoter was also identified as an important element for promoter activity in T cells; however, the responsible factor was unknown. In this report, we present data demonstrating that c-Myb binds to a Myb consensus site within the proximal promoter and is critical for its activity in T-lineage cells. We show that c-Myb can transactivate a RAG-2 promoter-reporter construct in cotransfection assays and that this transactivation depends on the proximal promoter Myb consensus site. By using a chromatin immunoprecipitation (ChIP) strategy, fractionation of chromatin with anti-c-Myb antibody specifically enriched endogenous RAG-2 promoter DNA sequences. DNase I genomic footprinting revealed that the c-Myb site is occupied in a tissue-specific fashion in vivo. Furthermore, an integrated RAG-2 promoter construct with mutations at the c-Myb site was not enriched in the ChIP assay, while a wild-type integrated promoter construct was enriched. Finally, this lack of binding of c-Myb to a chromosomally integrated mutant RAG-2 promoter construct in vivo was associated with a striking decrease in promoter activity. We conclude that c-Myb regulates the RAG-2 promoter in T cells by binding to this consensus c-Myb binding site.

Animals↗
PubMed2003

The murine G+C-rich promoter binding protein mGPBP is required for promoter-specific transcription.

The archetypal TATA-box deficient G+C-rich promoter of the murine adenosine deaminase gene (Ada) requires a 48-bp minimal self-sufficient promoter element (MSPE) for function. This MSPE was used to isolate a novel full-length cDNA clone that encodes a 66-kDa murine G+C-rich promoter binding protein (mGPBP). The mGPBP mRNAs are ubiquitously expressed as either 3.0- or 3.5-kb forms differing in 3' polyadenylation site usage. Purified recombinant mGPBP, in the absence of any other mammalian cofactors, binds specifically to both the murine Ada gene promoter's MSPE and the nonhomologous human Topo IIalpha gene's G+C-rich promoter. In situ binding assays, immunoprecipitation, and Western blot analyses demonstrated that mGPBP is a nuclear factor that can form complexes with TATA-binding protein, TFIIB, TFIIF, RNA polymerase II, and P300/CBP both in vitro and in intact cells. In cotransfection assays, increased mGPBP expression transactivated the murine Ada gene's promoter. Sequestering of GPBP present in HeLa cell nuclear extract by immunoabsorption completely and reversibly suppressed extract-dependent in vitro transcription from the murine Ada gene's G+C-rich promoter. However, transcription from the human Topo IIalpha gene's TATA box-containing G+C-rich promoter was only partially suppressed and the adenovirus major late gene's classical TATA box-dependent promoter is totally unaffected under identical assay conditions. These results implicate GPBP as a requisite G+C-rich promoter-specific transcription factor and provide a mechanistic basis for distinguishing transcription initiated at a TATA box-deficient G+C-rich promoter from that initiated at a TATA box-dependent promoter.

Adenosine Deaminase↗
PubMed1999

Health-promoting behaviours in public health: testing the health promotion model.

A health-promoting lifestyle encompasses far more than preventing disease and is characterised by behaviours that lead to optimal well-being, self-actualisation, and personal fulfillment. This study was undertaken to investigate the health-promoting lifestyles of employees working in local public health departments (n = 602) and to test two research hypotheses suggested by the Health Promotion Model. The first hypothesis posited that public health professionals would report significantly more favourable health-promoting behaviours than support staff employed in the same departments. The second hypothesis postulated that occupational discipline in public health would be a significant predictor of health-promoting behaviours. Health-promoting behaviours were measured by the Health-Promoting Lifestyle Profile (HPLP), a psychometrically-validated measure of overall health-promoting lifestyle and six dimensions of health-promoting behaviours, which comprise the instrument's subscales. The findings of the study tended to support the research hypotheses. Overall, public health professionals tended to report more favourable health-promoting behaviours than departmental support staff, and occupational discipline was a significant predictor of a health-promoting lifestyle. In general, public health nursing personnel reported the highest mean levels of health-promoting behaviours compared to the other occupational disciplines examined.

Adolescent↗
PubMed1994

Role of core promoter structure in assembly of the RNA polymerase II preinitiation complex. A common pathway for formation of preinitiation intermediates at many TATA and TATA-less promoters.

Efforts to understand the impact of core promoter architecture on the mechanism of transcription initiation by RNA polymerase II have been hampered by lack of well defined, reconstituted transcription systems responsive both to efficiently transcribed consensus and near consensus TATA box-containing promoters and to considerably weaker TATA-less promoters. In this report, we investigate the influence of core promoter structure on the mechanism of assembly of the RNA polymerase II preinitiation complex using a highly purified, holoTFIID-dependent transcription system that permits sensitive measurement of transcription initiation from a wide variety of TATA and TATA-less promoters in the absence of transactivators. A direct comparison of the requirements for formation of stable preinitiation intermediates at these promoters led to the discovery that, whereas holoTFIID binds avidly to the consensus TATA- and strong initiator-containing adenovirus major late (AdML) promoter to form the first stable intermediate on the pathway leading to formation of the complete preinitiation complex, it binds poorly not only to TATA-less promoters but also to promoters with consensus or near consensus TATA elements. With the exception of the AdML promoter, formation of stable preinitiation intermediates at each of the promoters tested was found to be strongly dependent on RNA polymerase II, holoTFIID, and TFIIB and was stimulated by TFIIF. Based on these observations, we suggest that RNA polymerase II assembles with many TATA and TATA-less promoters by a common pathway.

Base Sequence↗
PubMed1993

SP1 activates the MDR1 promoter through one of two distinct G-rich regions that modulate promoter activity.

To define sequences in the human multidrug resistance (MDR1) promoter that influence transcription, we measured the activity of MDR1 promoter constructs using luciferase as a reporter gene. Deletion of promoter sequences to -121 (relative to the transcription initiation site) had very little effect on promoter activity in transiently transfected cells. Further deletion to -88 increased activity about 4-fold, while deletion to -51 decreased activity about 10-fold. The data indicated that between -121 and -88 and between -73 and -51 were sequences that modulated promoter activity. Because these two regions contain G-rich sequences, which are important for function in other promoters, the effect of mutation of the G-rich regions (-110 to -103 and -61 to -43) on MDR1 promoter activity was measured. Mutation of the -110 G-box (-110 to -103) resulted in a 6-fold increase in promoter activity and inhibited the formation of a specific nuclear protein complex, suggesting that this region functions as a transcriptional "repressor" binding site in cycling cells. In contrast, mutation of the -50 G-box (-61 to -43) reduced promoter activity 5-fold. DNase-I footprinting and electrophoretic mobility shift analysis indicated that specific but distinct protein-DNA complexes formed at each of these two G-rich regions. The -50 G-box contains overlapping sites to which both SP1 and EGR-1 bind specifically. Co-transfection of MDR1 promoter constructs with SP1 into cells that lack SP1 (Drosophila Schneider 2 cells) activated equivalently both the wild type MDR1 promoter and a synthetic promoter containing the MDR1 -50 GC box and MDR1 initiator element.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Preferential primary-response gene expression in promotion-resistant versus promotion-sensitive JB6 cells.

The 12-O-tetradecanoylphorbol-13-acetate (TPA)-inducible sequence (TIS) genes are a set of primary response genes induced in Swiss 3T3 cells by TPA. They include three transcription factors, a prostaglandin synthase, and three proteins of unknown function. To ascertain which, if any TIS genes might be involved in tumor promotion, we examined the expression of these genes in response to tumor promoters in transformation promotion-sensitive (P+) and -resistant (P-) JB6 murine epidermal cells, a model used to identify events relevant to promotion. A subset of TIS genes (TIS1, TIS10, and TIS21) was preferentially induced by TPA in P-cells. In addition, TIS1 and TIS21 mRNAs were preferentially induced in P-cells by epidermal growth factor, another transformation promoter that distinguishes P+ from P-cells. TIS1 and TIS21 protein levels were also greater in TPA-treated P-cells than P+ cells. Forskolin, a cAMP-elevating anti-promoter, increased TPA-induced levels of TIS1, TIS10, and TIS21 mRNAs in P+ cells, ruling in potential roles for these genes in modulating tumor promotion. The anti-promoters fluocinolone acetonide, retinoic acid, and superoxide dismutase did not enhance TPA-induced levels of TIS1 and TIS21 mRNAs in P+ cells, suggesting that these inhibitors may act on other promotion-relevant genes. TIS1 encodes a member of the steroid receptor superfamily. TIS1 encodes a protein of unknown function with strong sequence similarity to BTG1, a proposed "anti-proliferative gene" (Rouault JP, Rimokh R, Tessa C, et al., EMBO J 11:1663-1670, 1992). Preferential induction by multiple promoters of these TIS genes in P-cells and enhancement of their induction in P+ cells by the anti-promoter forskolin make TIS1 and TIS21 candidates for promotion suppressor genes.

3T3 Cells↗
PubMed1980

Studies on the mechanism of skin tumor promotion: evidence for several stages in promotion.

The effects of nonpromoting and weakly promoting diterpenes on skin tumor promotion by 12-O-tetradecanoylphorbol 13-acetate (TPA) were investigated. When phorbol and phorbol 12,13-diacetate (both nonpromoting) were given simultaneously with TPA after 7,12-dimethylbenz[a]-anthracene (DMBA) initiation in female mice, they had no effect on TPA promotion. However, the nonpromoter 4-O-methyl-TPA and the weak promoter mezerein were found to inhibit TPA promotion in a dose-dependent manner when given simultaneously with TPA. Because mezerein was found to be an effective inhibitor of TPA promotion when given simultaneously and because it induces many biological responses similar to those to TPA, the capacity of mezerein to act as an incomplete promoter in a two-stage promotion protocol was also investigated. Twice-weekly applications of 1,2, or 5 mug of TPA for 2 weeks after DMBA initiation produced 0, 0, and 0.5 papilloma per mouse, respectively, at 20 weeks. When the twice-weekly applications of TPA for 2 weeks were followed by twice-weekly treatments with 2 mug of mezerein for 18 weeks, the number of papillomas per mouse was 2.2, 3.5, and 9.0, respectively. Twice-weekly applications of 2 mug of TPA for 2 weeks followed by twice-weekly treatments with 1, 2, or 4 mug of mezerein for 18 weeks produced 2.1, 3.5, and 6.8 papillomas per mouse, respectively, in DMBA-treated mice. Twice-weekly doses as high as 40 mug of 4-O-methyl-TPA were not effective in producing tumors when given after a limited treatment with TPA; however, 4-O-methyl-TPA had weak activity as a first-stage promoter. The results suggest that although mezerein by itself is a weak promoter and mimics TPA in many biochemical and morphological effects it is a potent second-stage promoter in a two-stage promotion regimen.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1999

Inactivation of MED-1 elements in the TATA-less, initiator-less mouse thymidylate synthase promoter has no effect on promoter strength or the complex pattern of transcriptional start sites.

The mouse thymidylate synthase (TS) promoter is a member of a family of promoters that lack a TATA box as well as an initiator element and that initiate transcription at many sites over a broad initiation window. An element (MED-1) downstream of the initiation window of almost all promoters of this family has been proposed to be important for promoter activity, as well as for multiple start site utilization. Two consensus MED-1 elements are located downstream of the initiation window of the TS promoter. To determine the role of the MED-1 elements in the TS promoter, one or both elements were inactivated by site-directed mutagenesis and the effects on promoter function were determined. We found that inactivation of the MED-1 elements had no measurable effect on promoter strength, the boundaries of the initiation window, or the pattern of transcriptional start sites. Furthermore, inactivation of the elements did not affect the ability of the TS promoter to direct S phase-specific expression of the gene in growth-stimulated cells. We conclude that the MED-1 element does not play a significant role in TS promoter function and therefore is not an essential component of all TATA-less promoters with complex transcriptional initiation patterns.

Animals↗
PubMed2005

Characterization of five evolutionary conserved regions of the human tyrosine hydroxylase (TH) promoter: implications for the engineering of a human TH minimal promoter assembled in a self-inactivating lentiviral vector system.

A DNA fragment of about 13 kb containing the human tyrosine hydroxylase (TH) promoter was previously isolated from a genomic DNA library and sequenced. The 11 kb from the transcription start of the human TH promoter was successively joined to the green fluorescent protein (GFP) to generate a transgenic mouse model. High levels of GFP expression could be observed in TH-positive cells of the Substantia nigra of embryonic and adult mice. Intriguingly, the sequence of the human TH promoter showed a low degree of homology with the mouse and rat TH promoters. In fact, comparative analysis of the sequences of human, rat, and mouse TH promoters revealed only five small regions of high homology. These five evolutionarily conserved regions were numbered in numeric progression from the 5' end of human TH promoter. In the present study, a panel of minimal human TH promoters was generated to analyze the transcriptional activity and specificity of gene expression conferred by the five conserved regions (CRs). The series of constructs was termed 250 bp and contained the first -194 bp of the human TH promoter immediately upstream of the transcription start, the first 35 bp the human TH messenger RNA leader, plus one or more of the five CRs. All the constructs were assembled in a self-inactivating form of the latest series of lentiviral vector system based on the human immunodeficiency virus type 1 (HIV-1). Lentiviral-mediated gene transfer was highly efficient for the in vitro transduction of human neuronal progenitor cells (hNPCs). Since a subset of hNPCs express TH following in vitro treatment with a mixture of differentiating agents, it was possible to assess specificity of expression for all the minimal human TH promoters. Overall, the successive addition of the five conserved regions produced a greater degree of specificity in induced TH-positive hNPCs, in particular after the addition of CRI (-8,917, -8,876). However, the human TH minimal promoters did not show any specificity for TH-positive differentiated mouse primary striatal and S. nigra cells, indicating a difference of TH gene regulation between the human and mouse systems. The human TH minimal promoters may provide the opportunity for the selection of TH-positive human embryonic and adult stem cells for brain transplantation experiments in animal models for Parkinson's disease.

Animals↗
PubMed2000

Construction of a promoter-rescue plasmid for Butyrivibrio fibrisolvens and its use in characterization of a flagellin promoter.

The Butyrivibrio fibrisolvens/Escherichia coli shuttle vector pBHerm has been modified to produce a plasmid (pBHE) that can be used for the identification and characterization of promoters in B. fibrisolvens. pBHE allows the insertion of a test promoter immediately upstream of a promoterless erythromycin resistance gene (ermAM). The efficacy of the pBHE plasmid in isolating and characterizing promoters was tested by inserting the flagellin gene (flaA) promoter from B. fibrisolvens OR77. Transcription of the ermAM gene from the flaA promoter was significantly higher than that observed when the ermAM gene was under the control of its own promoter. The flagelling gene of OR77 appears to be transcribed from two different promoters that produce transcripts initiating approximately 130 bp apart. Two mutant flaA promoter constructs, containing mutations in the -10 and -35 regions of either of the two putative promoter regions, showed drastic alterations in both the origin and amounts of the two transcripts produced. Mutations in either promoter affected transcription from both promoters, indicating that both regions contribute to gene expression.

DNA, Bacterial↗
PubMed1999

The bi-directional transcriptional promoters for the latency-relating transcripts of the pp38/pp24 mRNAs and the 1.8 kb-mRNA in the long inverted repeats of Marek's disease virus serotype 1 DNA are regulated by common promoter-specific enhancers.

In cell lines established from Marek's disease tumors, several viral transcripts are expressed and among them the products of pp38/pp24 mRNA and 1.8 kb-mRNA have been suggested to be involved in viral oncogenicity. The long inverted repeats of Marek's Disease virus serotype 1 (MDV1) genome contain closely located transcriptional promoters for phosphorylated protein pp38/pp24 and 1.8 kb-mRNA. These promoters initiate transcription in opposite directions and are separated only by a short enhancer region, which is likely to regulate both promoters simultaneously. We have analyzed the transcription activity of these promoters in MDV1 (Md5 strain) infected CEF by transient expression of CAT reporter genes and found that the promoters were in fact active in infected cells and the promoter for 1.8 kb-mRNA was more active than the pp38/pp24 promoter. Deletion analysis of the short enhancer region revealed that the 30 bp region overlapping the enhancer elements for 1.8 kb-mRNA was important for promoter activity for pp38/pp24. The gel shift analysis revealed that nuclear factor(s) actually bound to the overlapping 30 bp region. In addition, the activity of these promoters in infected cells varied with MDV strains. These results suggest that pp38/pp24 and 1.8 kb-mRNA promoters share a common regulatory sequence but a viral or a cellular factor(s) induced by viral infection regulates the promoter by distinct mechanisms.

Antigens, Viral↗
PubMed2006

Isolation of an enhancer from the rat tyrosine hydroxylase promoter that supports long-term, neuronal-specific expression from a neurofilament promoter, in a helper virus-free HSV-1 vector system.

Direct gene transfer into neurons, using a virus vector, has been used to study neuronal physiology and learning, and has potential for supporting gene therapy treatments for specific neurological diseases. Many of these applications require high-level, long-term recombinant gene expression, in forebrain neurons. We previously showed that addition of upstream sequences from the rat tyrosine hydroxylase (TH) promoter to a neurofilament heavy gene (NF-H) promoter supports long-term expression in forebrain neurons, from helper virus-free Herpes Simplex Virus (HSV-1) vectors. This element in the TH promoter satisfied the definition of an enhancer; it displayed activity at a distance from the basal promoter, and in both orientations. This enhancer supported physiological studies that required long-term expression; a modified neurofilament promoter, containing an insulator upstream of the TH-NFH promoter, supported expression in approximately 11,400 striatal neurons at 6 months after gene transfer, and expression for 7, 8, or 14 months, the longest times tested. In contrast, the NF-H promoter alone does not support long-term expression, indicating that the critical sequences are in the 6.3 kb fragment of the TH promoter. In this study, we performed a deletion analysis to identify the critical sequences in the TH promoter that support long-term expression. We localized these critical sequences to an approximately 320 bp fragment, and two subfragments of approximately 100 bp each. Vectors that contained each of these small fragments supported levels of long-term, neuronal-specific expression that were similar to the levels supported by a vector that contained the initial 6.3 kb fragment of the TH promoter. These small fragments of the TH promoter may benefit construction of vectors for physiological studies, and may support studies on the mechanism by which this enhancer supports long-term expression.

Animals↗
PubMed2003

In vitro studies of transcript initiation by Escherichia coli RNA polymerase. 1. RNA chain initiation, abortive initiation, and promoter escape at three bacteriophage promoters.

RNA chain initiation and promoter escape is the latter stage of transcription initiation. This stage is characterized by several well-defined biochemical events: synthesis and release of short RNA products ranging 2 to 15 nucleotides in length, release of the sigma subunit from the enzyme-promoter complex, and initial translocation of the polymerase away from the promoter. In this paper, we report the use of a steady-state transcription assay with [gamma-(32)P]ATP labeling to subject the RNA chain initiation-promoter escape reaction to quantitative analysis. The specific parameters we follow to describe the chain initiation-promoter escape process include the abortive and productive rates, the abortive probability, the abortive:productive ratio, and the maximal size of the abortive product. In this study, we measure these parameters for three bacteriophage promoters transcribed by Escherichia coli RNA polymerase: T7 A1, T5 N25, and T5 N25(antiDSR). Our studies show that all three promoters form substantial amounts of abortive products under all conditions we tested. However, each of the promoters shows distinct differences from the others when the various parameters are compared. At 100 microM NTP, in a 10 min reaction, the abortive and productive yields are 87 and 13%, respectively, for T7 A1; 97 and 3%, respectively, for T5 N25; and 99.4 and 0.6%, respectively, for T5 N25(antiDSR). These values correspond to approximately 7, 32, and 165 abortive transcripts per productive transcript for the three promoters, respectively. The yield of most of the abortive products is not affected by the elevated concentration of the NTP substrate corresponding to the next template-specified nucleotide; hence, abortive products are not normally formed through a simple process of "kinetic competition". Instead, formation of abortive products appears to be determined by intrinsic DNA signals embedded in the promoter recognition region and the initial transcribed sequence region of each promoter.

Adenosine Triphosphate↗
PubMed2001

Methylation in the p53 promoter is a supplementary route to breast carcinogenesis: correlation between CpG methylation in the p53 promoter and the mutation of the p53 gene in the progression from ductal carcinoma in situ to invasive ductal carcinoma.

Aberrant methylation in the CpG sites located in the promoter region of several tumor suppressor genes has been reported in various types of cancers. However, the methylation status of the p53 promoter has not been clearly determined and no information is available on its role in breast cancer. The aim of the study was to determine the presence and timing of the methylation of CpG sites in the p53 promoter, in the progression from ductal carcinoma in situ to invasive cancer. We also explored the correlation between the CpG methylation of the p53 promoter and p53 mutation during the progression of breast cancer. The corresponding lesions of both the invasive and noninvasive types were microdissected in paraffin-embedded tissue of 26 breast carcinomas. Bisulfite-modified DNA sequencing for methylation status in the p53 promoter was carried out, and double-strand DNA sequencing was performed in the promoter region and exons 4 to 9 of the p53 gene. CpG site methylation in the p53 promoter was detected in three cases (11.5%). Two noninvasive and three invasive lesions harbored CpG methylation in the p53 promoter. Methylations in more than one site were observed in three lesions, all of which contained methylation in two sites. The methylated CpG sites were located near the AP1 and YY-1 binding sites and at the YY-1 binding site. The p53 mutation was not found in the lesions where methylation in p53 promoter region was evident. In 16 cases (61.5%), neither methylation nor p53 mutation was detected. We conclude that the methylation in the p53 promoter region is found in the breast cancer irrespective of the status of invasion, and that the hypermethylation in the p53 promoter region is an alternative pathway to tumorigenesis where there is no p53 gene mutation.

Breast Neoplasms↗
PubMed2001

A novel method for the determination of basal gene expression of tissue-specific promoters: an analysis of prostate-specific promoters.

Because the toxicity of suicide gene therapeutics is directly related to basal promoter activity, we developed an assay to test for promoter "leakiness" using a diphtheria toxin mutant. Sequences of 15 prostate-specific gene promoter constructs were cloned in an expression plasmid (pBK; Stratagene, La Jolla, CA) backbone driving expression of an attenuated mutant of diphtheria toxin A (tox176). Low expression levels of the DT-tox176 result in significant protein synthesis inhibition reflected by a decreased expression of the luciferase activity of a simultaneously transfected CMV luciferase construct. ID50 (dose of plasmid with 50% luciferase inhibition) was calculated for each promoter construct in different cell lines. Highest transactivational activity (ID50 <75 ng) was found for the CMV promoter in all cell lines, which is in agreement with the dual luciferase assay findings. Unlike the dual luciferase findings, however, the DT-tox176 assay showed protein inhibition of CN65 (PSA promoter/enhancer) and PSE-hK2 (PSA enhancer and basal human kallikrein 2 promoter) in HEK293 and DLD cells indicating "leakiness" of these promoter constructs. Low basal promoter activity in nonprostate cell lines was found for the minimal PSA promoter, hK2, DD3, and OC promoters. The DT-tox176 assay can better predict basal promoter activity compared to less sensitive dual luciferase assay.

Biological Assay↗
PubMed1999

The maize mitochondrial cox2 gene has five promoters in two genomic regions, including a complex promoter consisting of seven overlapping units.

Plant mitochondrial genes are often transcribed into complex sets of RNAs, resulting from multiple initiation sites and processing steps. To elucidate the role of initiation in generating the more than 10 cox2 transcripts found in maize mitochondria, we surveyed sequences upstream of cox2 for active promoters. Because the cox2 coding region is immediately downstream of a 0.7-kb recombination repeat, cox2 is under the control of two different sets of potential expression signals. Using an in vitro transcription assay, we localized four promoters upstream of the coding region in the so-called master chromosome, and two promoters upstream of the coding region in the recombinant subgenome. Ribonuclease protection analysis of labeled primary transcripts confirmed that all but one of these promoters is active in vivo. Primer extension was used to identify the promoter sequences and initiation sites, which agree with the consensus established earlier for maize mitochondria. This study identified two unusual promoters, the core sequences of which were composed entirely of adenines and thymines, and one of which was a complex promoter consisting of seven overlapping units. Deletion mutagenesis of the complex promoter suggested that each of its units was recognized independently by RNA polymerase. While each active promoter fit the maize core consensus sequence YRTAT, not all such sequences surveyed supported initiation. We conclude that in vitro transcription is a powerful tool for locating mitochondrial promoters and that, in the case of cox2, promoter multiplicity contributes strongly to transcript complexity.

Base Sequence↗
PubMed2005

Weakening of the T7 promoter-polymerase interaction facilitates promoter release.

During transcription initiation, RNA polymerases retain interactions with their promoters until the RNA is extended to 8-13 nucleotides, at which point the polymerase releases the promoter and moves downstream. It has been shown that release of the T7 promoter is inhibited when the T7 RNA polymerase-promoter interaction is strengthened. Here we asked whether release would be facilitated when the T7 promoter-polymerase interaction is weakened by the introduction of promoter mutations known to reduce promoter activity. Using chemical and enzymatic probes to monitor the position of the polymerase as a function of RNA length, we found that promoter mutations upstream of -4 facilitated release of the polymerase from the promoter, but more downstream mutations did not have such effects. We also found that released complexes turn over more slowly than promoter-bound complexes, indicating that retention of promoter interactions contributes to the dissociation of short RNAs during initial transcription.

Bacteriophage T7↗