Upstream Promoter Elements Research Articles

Differential regulation of transcription of human 7 S K and 7 S L RNA genes.

Two functional human genes coding for 7 S RNA species K and L were analyzed for promoter requirements by in vitro transcription experiments with cytoplasmic S-100 extracts. Since accurate and efficient transcription of both genes is dependent on the presence of 5'-flanking sequences, hybrid genes representing crossover fusions between the 5' external control regions and the coding sequences of both genes were analyzed for their capacity to direct RNA synthesis in vitro. Differing results were obtained with both types of constructs. While the 5'-flanking L-7 S K gene fusion revealed no activity in the in vitro transcription assay, the 5'-flanking sequence of the 7 S K RNA gene did confer the ability for accurate in vitro transcription to the 7 S L coding sequence. However, a 5'-flanking L sequence element including the first 22 nucleotides of the 7 S L RNA coding sequence was active in promoting transcription of the 7 S K RNA gene. Together, these results demonstrated that the 7 S L promoter is located inside and outside the coding region, whereas the 7 S K RNA gene is exclusively controlled by an upstream promoter element.

Genetic profile of the transcriptional signals from the adenovirus major late promoter

Identical functional profiles were obtained for in vivo and in vitro transcription assays of more than 30 site-directed point mutants within the adenovirus major late promoter. The functional limits of the functional regions encompassing upstream promoter element are defined (−51 to −61), as well as a region around the transcription start site (−1 to +1), flanked by regions insensitive to sequence alterations.

Identification of three sequence-specific DNA-binding proteins which interact with the Rous sarcoma virus enhancer and upstream promoter elements.

Three avian nuclear proteins which bind to the Rous sarcoma virus long terminal repeat have been detected. Two of the proteins bind to sequences within the enhancer, and the third protein binds to a sequence spanning the enhancer and an upstream promoter region.

The COUP transcription factor binds to an upstream promoter element of the rat insulin II gene.

Band-shifting and DNase I-footprinting assays have been used to study the trans-acting factor(s) binding to an important promoter element (-53 to -46 relative to the transcription start) of the rat insulin II gene. A binding activity which footprints a region between -60 and -40 was found in both HIT, a hamster insulinoma cell line, and HeLa cells. A mutation within this region which drastically decreases promoter activity in vivo also greatly reduces binding activity in vitro. This binding activity was purified from HeLa cells and identified by competition and renaturation analyses as being the same as the COUP (chicken ovalbumin upstream promoter) transcription factor, a DNA-binding protein required for efficient transcription of the ovalbumin gene in vitro. Interestingly, the binding sequences of the COUP transcription factor in the ovalbumin and the insulin promoters have only limited similarities.

Saturation mutagenesis of a yeast his3 "TATA element": genetic evidence for a specific TATA-binding protein.

The yeast his3 promoter region contains two distinct classes of "TATA elements," constitutive (TC) and regulatory (TR), that are defined by their interactions with upstream promoter elements, selectivity of initiation sites, and chromatin structure. TC is localized between positions -83 and -53, and TR is localized between positions -55 and -35, regions in which there are several TATA-like sequences. In this study, we used saturation mutagenesis to examine the structural requirements of the his3 TR element necessary for transcriptional induction. To avoid the complications of redundant elements, the phenotypic analysis was carried out by using a gal-his3 hybrid promoter whose function depends on a short oligonucleotide containing the prospective his3 TR element. In this context, an oligonucleotide containing the sequence TATAAA is sufficient for TR function. However, 17 out of the 18 possible single-base substitutions and 9 out of 10 double mutations of this sequence abolish TR function. This strict sequence requirement for TR function strongly suggests that the TR element is a target site for a sequence-specific DNA-binding protein. Further, as the region encoding TC and promoters of certain other yeast genes do not contain a sequence that is compatible with TR function, we suggest that yeast cells contain multiple proteins with distinct sequence specificities that carry out a related "TATA function" and that yeast promoters can be divided into classes based on their downstream promoter elements.

Transcriptional activation of the Klebsiella pneumoniae nitrogenase promoter may involve DNA loop formation

Transcriptional activation of nitrogen fixation genes by NifA in Klebsiella pneumoniae requires an upstream NifA binding site. We now report that the introduction of half turns of the DNA helix into the DNA separating the upstream NifA binding site from the downstream promoter element of the nifH promoter decreases NifA-mediated activation to a greater extent than does the introduction of full helical turns. Reducing the spacing between the upstream and downstream elements of the nifH promoter also results in a promoter down phenotype. Introduction of a tight protein-binding site, the lac operator, between the upstream and downstream promoter elements did not render activation of the nifH promoter sensitive to occupancy of this site by the lac repressor. These findings indicate that NifA-mediated activation of transcription requires that NifA is bound upstream, and to the correct face of the DNA helix, in order to interact with downstream transcription factors. This implies that the interaction is brought about by the formation of a DNA loop between upstream and downstream promoter elements rather than by NifA sliding downstream.

Distinguishing between mechanisms of eukaryotic transcriptional activation with bacteriophage T7 RNA polymerase

To distinguish between mechanisms of eukaryotic transcriptional activation, we tested whether yeast upstream promoter elements can stimulate transcription by a heterologous transcription machinery, bacteriophage T7 RNA polymerase. The gal enhancer-like element recognized by GAL4 protein or the ded1 poly(dA-dT) element was placed upstream of the T7 promoter and his3 structural gene, and T7 RNA polymerase was produced in yeast cells. Under conditions where the gal element would normally be either activating or nonactivating, his3 transcription by T7 RNA polymerase was not stimulated above the level observed in the absence of any upstream element. In contrast, the ded1 poly(dA-dT) element stimulated transcription 7-fold, similar to the enhancement observed on the native ded1 promoter. Activation by the ded1 element thus may involve effects on the chromatin template that facilitate entry of the transcription machinery, whereas activation by the gal element may involve specific contacts between GAL4 and the transcriptional machinery.

Specific binding of TUF factor to upstream activation sites of yeast ribosomal protein genes.

Transcription activation of yeast ribosomal protein genes is mediated through homologous, 12-nucleotide-long and, in general, duplicated upstream promoter elements (HOMOL1 and RPG, referred to as UASrpg). As shown previously, a yeast protein factor, TUF, interacts specifically with these conserved boxes in the 5'-flanking sequences of the elongation factor genes TEF1 and TEF2 and the ribosomal protein gene RP51A. We have now extended our studies of TUF-UASrpg binding by analysing--using footprinting and gel electrophoretic retardation techniques--the genes encoding the ribosomal proteins L25, rp28 (both copy genes), S24 + L46 and S33. Most, but not all, conserved sequence elements occurring in front of these genes, turned out to represent binding sites for the same factor, TUF. The two functionally important boxes that are found in a tandem arrangement (a characteristic of many rp genes) upstream of the L25 gene are indistinguishable in their factor binding specificity. Large differences were shown to exist in the affinity of the TUF factor for the various individual boxes and in the half-life of the protein-DNA complexes. No binding cooperativity could be demonstrated on adjacent sites on L25 or RP51A promoters. Based on binding data, the UASrpg sequence ACACCCATACAT appears to be the one recognized most efficiently by the TUF factor. Previously, no conserved box was found in front of the gene encoding S33. Nevertheless, complex formation with the protein fraction used was observed in the upstream region of the S33 gene. Competition experiments disclosed the existence of an additional binding component, distinct from TUF. This component may possibly regulate a subset of genes for the translational apparatus.

Structure and expression of the Saccharomyces cerevisiae CRY1 gene: a highly conserved ribosomal protein gene.

The Saccharomyces cerevisiae CRY1 gene encodes ribosomal protein rp59, a component of the 40S ribosomal subunit. Mutations in CRY1 can confer resistance to the alkaloid cryptopleurine, an inhibitor of the elongation step of translation. The nucleotide sequence of the cloned CRY1 gene was determined. The predicted amino acid sequence shows that CRY1 encodes a 14,561-dalton polypeptide that has 88% amino acid sequence homology to the hamster or human S14 ribosomal protein responsible for emetine resistance and 45% homology to Escherichia coli ribosomal protein S11. Analysis of the DNA sequences upstream from CRY1 revealed the presence of three sequences, HOMOL1 (consensus, A/TACATCC/TG/ATA/GCA), RPG (consensus, ACCCA/GTACATT/CT/A), and a thymine-rich sequence, found upstream of more than 20 other cloned yeast genes encoding components of the translational apparatus. We exploited the ability to assay the expression of CRY1 in vivo by using the cryptopleurine resistance phenotype to demonstrate that these three consensus sequences are necessary for the transcription of CRY1. We previously showed that the upstream promoter element of the yeast RP39A gene consists of these identical sequence motifs. Therefore, we suggest that these three sequences define a consensus promoter element for the genes encoding the yeast translational apparatus. CRY1 is one of several hundred yeast genes, including ribosomal protein genes, whose expression is transiently decreased 10-fold upon heat shock. We found that the HOMOL1 and RPG consensus sequences are not necessary for the heat shock response of CRY1.

Structure and Expression of the Saccharomyces Cerevisiae CRY1 Gene: a Highly Conserved Ribosomal Protein Gene

The Saccharomyces cerevisiae CRY1 gene encodes ribosomal protein rp59, a component of the 40S ribosomal subunit. Mutations in CRY1 can confer resistance to the alkaloid cryptopleurine, an inhibitor of the elongation step of translation. The nucleotide sequence of the cloned CRY1 gene was determined. The predicted amino acid sequence shows that CRY1 encodes a 14,561-dalton polypeptide that has 88% amino acid sequence homology to the hamster or human S14 ribosomal protein responsible for emetine resistance and 45% homology to Escherichia coli ribosomal protein S11. Analysis of the DNA sequences upstream from CRY1 revealed the presence of three sequences, HOMOL1 (consensus, A/TACATCC/TG/ATA/GCA), RPG (consensus, ACCCA/GTACATT/CT/A), and a thymine-rich sequence, found upstream of more than 20 other cloned yeast genes encoding components of the translational apparatus. We exploited the ability to assay the expression of CRY1 in vivo by using the cryptopleurine resistance phenotype to demonstrate that these three consensus sequences are necessary for the transcription of CRY1. We previously showed that the upstream promoter element of the yeast RP39A gene consists of these identical sequence motifs. Therefore, we suggest that these three sequences define a consensus promoter element for the genes encoding the yeast translational apparatus. CRY1 is one of several hundred yeast genes, including ribosomal protein genes, whose expression is transiently decreased 10-fold upon heat shock. We found that the HOMOL1 and RPG consensus sequences are not necessary for the heat shock response of CRY1.

The ovarian, ecdysterone, and heat-shock-responsive promoters of the Drosophila melanogaster hsp27 gene react very differently to perturbations of DNA sequence.

The effect of various types of DNA sequence alterations on the activity of the ovarian, ecdysterone, and heat-shock-responsive promoters of the Drosophila melanogaster hsp27 gene was studied by P element-mediated germ line transformation. Regions of DNA required for proper expression of the gene under these different conditions were identified. Wild-type levels of transcription during oogenesis are dependent on two elements respectively located within a 64-base-pair (bp) fragment in the transcribed untranslated region and between -227 and -958 bp upstream of the transcription start site. This ovarian expression is particularly sensitive to both chromosomal position effects and an increased distance between the distal upstream promoter element and the TATAA homology. The ecdysterone-mediated expression during metamorphosis is dependent on a 145-bp domain including the TATAA box and additional upstream sequences that augment transcription by two- to five-fold. Finally, sequences necessary for heat shock expression are located much further upstream from hsp27 than those previously found for hsp70, although basal expression was correlated with the presence of more proximal heat shock consensus sequences.

The Ovarian, Ecdysterone, and Heat-Shock-Responsive Promoters of the Drosophila melanogaster hsp27 Gene React Very Differently to Perturbations of DNA Sequence

The effect of various types of DNA sequence alterations on the activity of the ovarian, ecdysterone, and heat-shock-responsive promoters of the Drosophila melanogaster hsp27 gene was studied by P element-mediated germ line transformation. Regions of DNA required for proper expression of the gene under these different conditions were identified. Wild-type levels of transcription during oogenesis are dependent on two elements respectively located within a 64-base-pair (bp) fragment in the transcribed untranslated region and between -227 and -958 bp upstream of the transcription start site. This ovarian expression is particularly sensitive to both chromosomal position effects and an increased distance between the distal upstream promoter element and the TATAA homology. The ecdysterone-mediated expression during metamorphosis is dependent on a 145-bp domain including the TATAA box and additional upstream sequences that augment transcription by two- to five-fold. Finally, sequences necessary for heat shock expression are located much further upstream from hsp27 than those previously found for hsp70, although basal expression was correlated with the presence of more proximal heat shock consensus sequences.

Regulation of mRNA accumulation by a human immunodeficiency virus trans-activator protein

HIV LTR-directed expression is markedly stimulated in trans by coexpression of a region of the HIV genome encoding a portion of the tat reading frame. Transient expression assay analysis reveals that trans-activation of LTR-directed expression results primarily from an increase in mRNA accumulation. Deletion analysis of the LTR indicates that upstream promoter and enhancer elements are dispensible for trans-activation, while sequences 3′ of the RNA start site displaying strict orientation and position dependence are required. These sequences, contained in the 5′ leader of all HIV transcripts, form a stable stem-loop structure with twofold symmetry in the cognate mRNA. Analysis of mutations in the trans-acting region demonstrates that the trans-activator is the protein product of the tat gene, identified biochemically in HIV-infected and transfected cells as an M r 15,000 polypeptide. We discuss possible mechanisms whereby the interaction of p15 tat with the dyad element promotes the accumulation of LTR-directed mRNA.

Maize Adh-1 promoter sequences control anaerobic regulation: addition of upstream promoter elements from constitutive genes is necessary for expression in tobacco

The promoter region of a maize alcohol dehydrogenase gene (Adh-1) was linked to a reporter gene encoding chloramphenicol acetyl transferase (CAT) and transformed stably into tobacco cells using T-DNA vectors. No CAT enzyme activity could be detected in transgenic tobacco plants unless upstream promoter elements from the octopine synthase gene or the cauliflower mosaic virus 35S promoter were supplied in addition to the maize promoter region. CAT enzyme activity and transcription of the chimaeric gene were then readily detected after anaerobic induction. The first 247 bp upstream of the translation initiation codon of the maize Adh-1 gene were sufficient to impose anaerobic regulation on the hybrid gene and S1 nuclease mapping confirmed mRNA initiation is from the normal maize Adh-1 transcription start point.

Xenopus tropicalis U6 snRNA genes transcribed by Pol III contain the upstream promoter elements used by Pol II dependent U snRNA genes.

We have cloned and sequenced a 977bp DNA fragment, pXTU6-2, that represents the transcription unit for a Xenopus tropicalis U6 RNA gene. This basic repeating unit is reiterated ca.500-fold per haploid genome. Oocyte injections of pXTU6-2 led to the transcription of a mature-sized U6 RNA that, however, lacked internal 2'-O-methylations. These posttranscriptional modifications of U6 RNA might be cytoplasmic and could require its association with U4 RNA to be accomplished. The low alpha- amanitin sensitivity of U6 RNA synthesis in oocytes suggested that U6 RNA is transcribed by RNA polymerase III, consistent with features of the U6 RNA molecule which also contains a Box A- like intragenic control region. Inspection of X. tropicalis, mouse and human U6 DNA upstream sequences revealed the presence of a TATA box as well as of the proximal and enhancer (octamer motif) elements contained in snRNA genes transcribed by RNA polymerase II. We propose that U6 RNAs are synthesized by a specialized transcription complex consisting of RNA polymerase III and transcription factors, some of which are very likely shared with RNA polymerase II promoters.

Constitutive and inducible Saccharomyces cerevisiae promoters: evidence for two distinct molecular mechanisms.

his3 and pet56 are adjacent Saccharomyces cerevisiae genes that are transcribed in opposite directions from initiation sites that are separated by 200 base pairs. Under normal growth conditions, in which his3 and pet56 are transcribed at similar basal levels, a poly(dA-dT) sequence located between the genes serves as the upstream promoter element for both. In contrast, his3 but not pet56 transcription is induced during conditions of amino acid starvation, even though the critical regulatory site is located upstream of both respective TATA regions. Moreover, only one of the two normal his3 initiation sites is subject to induction. From genetic and biochemical evidence, I suggest that the his3-pet56 intergenic region contains constitutive and inducible promoters with different properties. In particular, two classes of TATA elements, constitutive (Tc) and regulatory (Tr), can be distinguished by their ability to respond to upstream regulatory elements, by their effects on the selection of initiation sites, and by their physical structure in nuclear chromatin. Constitutive and inducible his3 transcription is mediated by distinct promoters representing each class, whereas pet56 transcription is mediated by a constitutive promoter. Molecular mechanisms for these different kinds of S. cerevisiae promoters are proposed.

Constitutive and inducible Saccharomyces cerevisiae promoters: evidence for two distinct molecular mechanisms.

his3 and pet56 are adjacent Saccharomyces cerevisiae genes that are transcribed in opposite directions from initiation sites that are separated by 200 base pairs. Under normal growth conditions, in which his3 and pet56 are transcribed at similar basal levels, a poly(dA-dT) sequence located between the genes serves as the upstream promoter element for both. In contrast, his3 but not pet56 transcription is induced during conditions of amino acid starvation, even though the critical regulatory site is located upstream of both respective TATA regions. Moreover, only one of the two normal his3 initiation sites is subject to induction. From genetic and biochemical evidence, I suggest that the his3-pet56 intergenic region contains constitutive and inducible promoters with different properties. In particular, two classes of TATA elements, constitutive (Tc) and regulatory (Tr), can be distinguished by their ability to respond to upstream regulatory elements, by their effects on the selection of initiation sites, and by their physical structure in nuclear chromatin. Constitutive and inducible his3 transcription is mediated by distinct promoters representing each class, whereas pet56 transcription is mediated by a constitutive promoter. Molecular mechanisms for these different kinds of S. cerevisiae promoters are proposed.

Binding of polyomavirus large T antigen to the human hsp70 promoter is not required for trans activation.

Polyomavirus large T antigen binds to two sites located between positions -110 and -170 of a human heat shock protein 70 (hsp70) promoter. Methylation interference studies show that binding for each site is determined by two GPuGGC pentanucleotide sequences. The specificity of this binding interaction is similar to that observed for large T binding to the viral genome. The existence of sequences that bind a viral protein in a cellular promoter raises the possibility that these sequences play a role in gene expression in an uninfected cell. We show that hsp70 large T antigen binding site 1 is capable of functioning as an upstream promoter element in cells that do not contain any viral T antigen. Genetic analysis of this effect suggests that a cellular factor exists that has a binding specificity that overlaps but is not identical to that of polyomavirus large T antigen. To determine whether binding of polyomavirus large T antigen can regulate expression of the intact human hsp70 promoter, we have introduced the promoter into mouse cells with plasmids that express the polyomavirus early proteins. These proteins stimulate the level of correctly initiated hsp70 transcripts, but surprisingly the degree of stimulation remains unchanged for promoter constructs in which the large T antigen binding sites have been deleted. These observations suggest that trans activation of the hsp70 promoter by the polyomavirus early proteins occurs through protein-protein interactions and not through sequence-specific DNA binding.

Binding of polyomavirus large T antigen to the human hsp70 promoter is not required for trans activation.

Polyomavirus large T antigen binds to two sites located between positions -110 and -170 of a human heat shock protein 70 (hsp70) promoter. Methylation interference studies show that binding for each site is determined by two GPuGGC pentanucleotide sequences. The specificity of this binding interaction is similar to that observed for large T binding to the viral genome. The existence of sequences that bind a viral protein in a cellular promoter raises the possibility that these sequences play a role in gene expression in an uninfected cell. We show that hsp70 large T antigen binding site 1 is capable of functioning as an upstream promoter element in cells that do not contain any viral T antigen. Genetic analysis of this effect suggests that a cellular factor exists that has a binding specificity that overlaps but is not identical to that of polyomavirus large T antigen. To determine whether binding of polyomavirus large T antigen can regulate expression of the intact human hsp70 promoter, we have introduced the promoter into mouse cells with plasmids that express the polyomavirus early proteins. These proteins stimulate the level of correctly initiated hsp70 transcripts, but surprisingly the degree of stimulation remains unchanged for promoter constructs in which the large T antigen binding sites have been deleted. These observations suggest that trans activation of the hsp70 promoter by the polyomavirus early proteins occurs through protein-protein interactions and not through sequence-specific DNA binding.

Heat shock regulatory elements function as an inducible enhancer in the xenopus hsp70 gene and when linked to a heterologous promoter

The Xenopus hsp70 promoter contains three copies of the consensus heat shock element (HSE) between positions −260 and −100. When the gene is transfected into mammalian cells, maximal heat-induced expression requires two HSEs in addition to a CCAAT box located next to the TATA box. The HSE-containing region can be separated from the CCAAT/TATA region without affecting expression of the gene, and it can enhance transcription of a linked β-globin gene upon heat shock. It thus has the properties of a heat-inducible enhancer. Such an enhancer can also be generated by duplication of HSE sequences from the Drosophila hsp70 promoter, which were previously identified as an upstream promoter element and are known to bind a purified heat shock transcription factor in vitro.