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PubMed1988

Identification of an upstream activation site in the pyruvate decarboxylase structural gene (PDC1) of Saccharomyces cerevisiae.

The upstream region of the Saccharomyces cerevisiae pyruvate decarboxylase structural gene, PDC1, has been isolated and fused to the indicator gene Escherichia coli lacZ. 1.2 kb of the upstream region has been sequenced. The PDC1-lacZ fusion has been integrated at the ura3-52 locus in the yeast genome, and has a basal level of expression on ethanol. On glucose media this level is increased 30-50 fold. An upstream activation site, UASpdc, between 793 and 535 bp upstream from the ATG of PDC1, which mediates the response to glucose has been identified by deletion analysis. The UASpdc contains a consensus RPG box, originally identified in ribosomal protein genes (Leer et al. 1985). The function of UASpdc is independent of distance from the ATG. There is also an upstream repressing sequence located between 535 and 385 bp upstream from the translational start of PDC1.

Base Sequence↗
PubMed2000

An Eel Cytochrome P450 1A Gene Having No XRE Sequences in Its 5' Upstream Region of 1600 bp.

The gene family cytochrome P450 (CYP) 1A is ubiquitous among animals, and CYP1A1 genes have been identified in teleost and mammals. We isolated another CYP1A gene, which is inducibly expressed by exposure to 3-methylcholanthrene, from the genomic library of the eel (Anguilla japonica). The genomic clone obtained, approximately 17,500 bp in length, contained the structural gene of the CYP1A and a 5' upstream region of about 1600 bp. Sequence analysis of the 5' upstream region and the first and second exons revealed that the initiation codon was in the second exon, as in the CYP1A genes reported for mammals and teleosts. CAAT and TATA boxes were found 51 and 29 bp upstream from the transcriptional start site, respectively. Unlike the CYP1A1 genes of eel and other animals, we found no xenobiotic responsive element (XRE) core sequences in the 5' upstream region studied (1631 bp) or in the first intron, whereas three peculiar regions, each composed of multiple repeats of the trinucleotide TAA, were found between 824 and 1356 bp upstream from the transcriptional start site. Absence of XRE core sequences and presence of the multiple repeats in the 5' upstream region sequenced may suggest that the expression mechanism of the eel CYP1A gene is somewhat different from the mechanisms of other reported CYP1A genes.

Journal Article↗
PubMed1988

The upstream limit of nuclease-sensitive chromatin in Dictyostelium rRNA genes neighbors a topoisomerase I-like cluster.

In the chromatin of Dictyostelium ribosomal RNA (rRNA) genes, the coding and upstream flanking regions are sensitive to endonucleases. This sensitivity stops about 2.3 x 10(3) bases upstream from the transcription start, at a point we call the structural boundary. Upstream from the boundary an 850 base-pair region is strongly protected against micrococcal nuclease cleavage, particularly in rapidly transcribing vegetative cells, and upstream from this the pattern of nuclease protection suggests that positioned nucleosomes are present. On the gene side of the structural boundary nucleosomes are known to be absent in vegetative cells but present in differentiating slug cells where the rRNA synthesis rate is lower. We show that in slugs these nucleosomes are randomly distributed, in contrast to those upstream from the boundary. Close to the gene side of the boundary is a duplication of the putative promoter located 29 base-pairs distant from four clustered topoisomerase I recognition sequences, which are cleaved by endogenous topoisomerase I-like activity. An additional topoisomerase I recognition sequence found upstream from the structural boundary is not cleaved in chromatin. The possible significance of these sequences and structures in transcription is discussed.

Binding Sites↗
PubMed1991

Multiple sets of adjacent mu E1 and oct-1 binding sites upstream of the pseudorabies virus immediate-early gene promoter.

Binding of cellular proteins to specific motifs in the promoter region of the immediate-early gene of pseudorabies virus was studied. The region was dissected into several portions that were used with HeLa cell nuclear proteins in mobility shift assays, DNase I footprinting, and a methylation interference assay. Close to the transcription start site (nucleotide + 1) are a TATA-box (-26 to -29), an Sp 1-binding motif (GGGGCGGGC) (-45 to -54), a CCAAT motif (-66 to -70), and, further upstream, an NF-microE1-binding site (AAGATGGC) (-161 to -168). Binding of a protein to the Sp1 site was demonstrated. Competition experiments show that the CCAAT motif might bind the NF-microE1 factor, rather than any of the known CCAAT-specific factors. Four domains were identified further upstream (nucleotides -200 to -500) from this promoter, each of which contained closely associated motifs where cellular transcription factors NF-microE1 and oct-1 could bind. These domains comprise what we call the upstream element. The orientation of the four NF-microE1 motifs in the upstream element is opposite to the orientation of the two NF-microE1 motifs located closer to the transcription start site. Transient expression of reporter genes was used to study the activity of the upstream element after transfection into 3T3 and HeLa cells. The upstream element was necessary for efficient expression of the pseudorabies virus immediate-early gene and increased somewhat the efficiency of the herpes simplex virus thymidine kinase promoter.

Base Sequence↗
PubMed1996

Cell type-specific expression of the mouse peripherin gene requires both upstream and intragenic sequences in transgenic mouse embryos.

Peripherin is a neuron-specific type III intermediate filament protein expressed in well-defined populations of neurons projecting towards peripheral targets. To investigate the molecular mechanisms by which a gene is expressed in a specific subset of neurons, we used a transgenic approach in order to define peripherin gene sequences that are necessary for cell-type specific expression. Transgenic mice carrying different various genomic regions of the mouse peripherin gene fused to the Escherichia coli lacZ reporter gene were generated. We used three different peripherin/lacZ constructs containing either 5.8 kb upstream sequences, or both 5.8 kb upstream and 1.1 kb intragenic sequences, or 1.1 kb intragenic sequences associated with an heterologous promoter. Analysis of lacZ gene expression in transgenic mouse embryos showed that cell type-specific expression of the mouse peripherin gene requires both upstream and intragenic sequences. Analysis of transgenic mouse lines expressing the construct containing both upstream and intragenic sequences showed that this transgene contains all regulatory elements essential for both spatial and temporal expression of the mouse peripherin gene during embryogenesis. Furthermore, lacZ+ positive cells isolated from these transgenic lines by fluorescence-activated cell sorting (FACS) can be stained with a peripherin antibody, demonstrating that the transgene containing both upstream and intragenic sequences is expressed in peripherin neurons. These mouse peripherin upstream and intragenic sequences can now be used to identify cis-acting regulatory elements and transcription factors involved in peripherin gene regulation.

Animals↗
PubMed1995

Upstream and intron regulatory regions for expression of the rat neuron-specific enolase gene.

Neuron-specific enolase (NSE) occurs in mature neurons and paraneurons. We have isolated the genomic clone coding for rat NSE and clarified its gene structure. In order to analyze the regulatory sequence in the 5'-upstream region and introns, we carried out transient expression experiments of NSE genomic DNA fragments fused to chloramphenicol acetyltransferase (CAT) gene which were transfected into several cultured cells. The used cells were primary cultured rat neurons, PC12, neuroblastoma 35, neuroblastoma 103, C6, primary cultured rat glial cells and HeLa cells. The promoter sequence (190 bp) upstream to the transcription initiation site was important in the expression of CAT gene in these cells. From the experiments with external and internal deletion mutants of the fusion gene, the cis-acting regulatory region responsible for the enhanced expression of the CAT activity in the primary cultured neuron and PC12 cells was found to be localized at upstream 500 bp sequence of the intron 1 and 1.5 kbp upstream sequence of the transcription initiation site. In the upstream important sequences, there were the nearest sequences for AP-1 binding motif, AP-2 binding element, SP-1 binding sequence, cAMP response element, half site of glucocorticoid receptor (GRE) binding sequence, half site of thyroid hormor receptor (TR) or retinoic acid receptor (RAR) binding sequence and MTF-1 binding sequence. Furthermore, Octamer-6 binding motifs also were found. In the intron 1, 5' end upstream 50 bp and downstream 100 bp were the most important sequences. We found the nearest sequences for cAMP response element, E2F binding sequence, early growth response (EGR)-1 binding motif, half site of TCF-1 binding sequence and a neuron-specific element-like sequence in the intron 1.

Animals↗
PubMed1996

Isolation, characterization and in vivo analysis of the murine calbindin-D28K upstream regulatory region.

The genomic locus containing the murine calbindin-D28K gene has been isolated and partially characterized. Genomic cloning revealed an exon/intron chromosomal structure very similar to the avian gene previously described. The ability of the calbindin-D28K upstream region to direct cell-specific expression was tested in vivo. Varying lengths of upstream sequence were used to drive expression of lacZ in transgenic mice. Characterization of 23 transgenic mouse lines revealed that even as much as 3.0 kb of upstream sequence was unable to direct expression independently of integration site effects, suggesting the absence of important elements. Despite the small number of expressing transgenic lines and the great variability, there was a tendency of cell specificity of transgene expression exhibited in distinct brain regions. In the cerebellum, Purkinje cell-specific expression was observed with the shortest (1.0 kb) upstream sequence tested. Specificity of transgene expression in Purkinje cells was abolished with longer portions of upstream sequence. The same observation was made for transgene expression in granule cells of the dentate gyrus, while the opposite effect was observed for expression in CA1 hippocampal cells. The absence of any transgenic lines exhibiting appropriate transgene expression in the kidney suggested that the VDREs described previously for the murine calbindin gene are not sufficient to direct kidney expression in vivo. It is concluded that 3.0 kb of calbindin upstream sequence includes the regulatory elements dictating a portion of cell-specificity in the CNS of transgenic mice, albeit lacking regions that allow expression independently of chromosomal effects.

Animals↗
PubMed2006

The role of risk stratification in the decision to provide upstream versus selective glycoprotein IIb/IIIa inhibitors for acute coronary syndromes: a cost-effectiveness analysis.

OBJECTIVES: We endeavored to determine under what conditions a strategy of upstream use of small molecule platelet glycoprotein (GP) IIb/IIIa inhibitors for all acute coronary syndromes (ACS) patients is cost effective compared to that of selective use of abciximab in only those patients requiring percutaneous coronary intervention (PCI). BACKGROUND: Small molecule GP IIb/IIIa inhibitors have shown benefit in ACS, but abciximab, the more expensive GP IIb/IIIa inhibitor, may be more effective during PCI. However, abciximab does not have proven efficacy in medical management. No prior study has attempted to balance these competing benefits. METHODS: A decision analysis was performed to examine two strategies: 1) treat all ACS patients upstream with a small molecule GP IIb/IIIa inhibitor and continue through medical management and PCI, if performed; or 2) wait, and selectively use abciximab only in patients who ultimately undergo PCI. Applicable randomized controlled trial data were used for the principal analysis. RESULTS: The strategy of upstream use of a small molecule GP IIb/IIIa inhibitor was superior to selective use, and economically acceptable, with a cost-effectiveness ratio of 18,000 dollars per year of life gained. The superiority of the upstream use strategy persisted over the majority of sensitivity analyses. When stratified by risk according to Thrombolysis in Myocardial Infarction risk score, a strategy of upstream use was only cost effective in those patients with moderate or high risk. CONCLUSIONS: Upstream use of small molecule GP IIb/IIIa inhibition in ACS patients with moderate or high risk for cardiovascular events is a cost-effective approach that should be considered in this subset of patients.

Abciximab↗
PubMed1984

Conserved structural features are found upstream from the three co-ordinately regulated discoidin I genes of Dictyostelium discoideum.

The discoidin I genes of Dictyostelium form a small, co-ordinately regulated multigene family. We have sequenced and compared the upstream regions of the DiscI-alpha, -beta and -gamma genes. For the most part the upstream regions of the three genes are non-homologous. The upstream sequences of the beta and gamma genes are exceedingly A + T-rich, while those of the alpha gene are less so. All three genes have a relatively G + C-rich region 20 to 40 base-pairs in length, found approximately 200 base-pairs 5' to the messenger RNA start site. This G + C-rich region 5' to the beta and gamma genes is flanked by short inverted repeats. Within this region, there is an 11 base-pair exact homology between the alpha and gamma genes, and a less perfect homology between these genes and the beta gene. The homology is flanked at a short distance by interspersed G and T residues. The gamma gene is greater than 90% A + T for greater than 800 base-pairs upstream. Further upstream there is a G + C-rich region that is also found inverted approximately 3.5 X 10(3) base-pairs away. The gamma and beta genes are tandemly linked, and the entire approximately 500 base-pair intergene region between the 3' end of the gamma gene and the 5' end of the beta gene is A + T-rich (approximately 90%) with the exception of the homology region 5' to the gamma gene. We demonstrate also the presence of a discoidin I pseudogene fragment having only 139 base-pairs of discoidin homology with greater than 8% mismatch. It is flanked upstream by five 39 base-pair G + C-rich repeats, and downstream by sequences that are extremely A + T-rich. We discuss the possible significance of the conserved G + C-rich structures on discoidin I gene expression.

Base Sequence↗
PubMed1997

The normally silent sigma54 promoters upstream of the pilE genes of both Neisseria gonorrhoeae and Neisseria meningitidis are functional when transferred to Pseudomonas aeruginosa.

The pilE gene encodes the pilin subunit in Neisseria gonorrhoeae and Neisseria meningitidis. Transcriptional analysis of promoters upstream of pilE in N. gonorrhoeae has been described previously (Fyfe et al. (1995) J. Bacteriol. 177, 3781-3787). Transcription from the sigma54-dependent promoter P3 was detected in Pseudomonas aeruginosa. Here we show that this transcription is dependent on the P. aeruginosa transcriptional activator PilR, and a specific upstream sequence with a high degree of similarity to the PilR-binding site found upstream of the P. aeruginosa pilin gene. This implies there is an upstream activator site (UAS) present 5' of pilE. Sequencing upstream of the N. meningitidis MC58 c2 pilE gene shows this region to be very similar to that in N. gonorrhoeae. P3 and the UAS are conserved, although insertions were noted on either side of the UAS. Transcriptional analysis has shown that the N. meningitidis P3 promoter is used in P. aeruginosa, provided PilR and an upstream region that includes sequence similar to the UAS are present. Transcription from the N. meningitidis PpilE is stronger than from the N. gonorrhoeae equivalent. N. meningitidis uses the sigma70 promoter P1 to transcribe pilE.

Bacterial Adhesion↗
PubMed1998

Modifications in protein binding to upstream sequences of the sea urchin cytoplasmic actin gene CyIIa in comparison to its linked neighbors, CyI and CyIIb.

The sequences corresponding to regions upstream of the ATG and transcription start site of the CyIIa cytoplasmic actin gene of the sea urchin Strongylocentrotus purpuratus were determined and compared to the genomically linked CyI and CyIIb actin genes. Sites of protein-DNA interaction in the CyIIa upstream sequences were identified by DNase I footprinting. The similarity between CyIIa and CyI (and CyIIb) upstream sequences was limited and included a consensus octamer sequence, serum response element (SRE) and some short sequences within the proximal promoter region. The octamer sequence was found to bind protein. A single DNase I hypersensitive site was detected within the SRE and to two flanking nucleotides, but otherwise, the SRE did not appear to be protected. This is in contrast to strong protein binding to the CyIIb SRE. A region in the CyIIa gene with limited identity to the functionally significant protein binding site D in CyI also did not bind protein. Four additional sites of protein-DNA interaction were identified in CyIIa upstream sequences. One of these is similar to a protein binding site previously located in the CyI upstream sequences, whereas the others appear to be unique. These data indicate that the CyIIa upstream sequences differ extensively from those of CyI. The pattern of CyIIa expression is likely a consequence of these alternations in DNA sequence and protein-DNA interactions.

Actins↗
PubMed2002

Analysis of regulatory functions for the region located upstream from the latency-associated transcript (LAT) promoter of pseudorabies virus in cultured cells.

The latency-associated transcript (LAT) promoter of pseudorabies virus (PrV) is unique among the many promoters of the viral genome in that it remains active during the latent state. The regulatory mechanism of PrV LAT gene expression is complex and different between latency and lytic infection of cultured cells. Although two different sequences, LAP1 and LAP2, are thought to be involved in LAT gene expression, the function of the upstream region of the LAT promoter (LAP1 and LAP2) remains an enigma, even in cultured cells. To analyze the function of the upstream region, it is necessary to examine the effects of the upstream sequence on LAT gene expression in the absence of other viral proteins. Transient expression assays were performed by employing a series of reporter plasmids in which various sequences upstream of the LAT promoter (from nucleotide positions -592 to +423 relative to the transcriptional start site of the large latency transcript (LLT)) were linked to the chloramphenicol acetyltransferase (CAT) gene in cells of neuronal and non-neuronal origin. We identified a region (from nucleotide positions -3606 to -1386) that was capable of repressing the LAT promoter activity in Vero cells by analyzing CAT gene expression of the series of reporter plasmids. This effect was not observed in Neuro-2a cells. We have also shown that the LAT promoter activity of the reporter plasmid containing the upstream region was repressed by the immediate-early gene product IE180 in Vero cells, but not in Neuro-2a cells. These results suggest that the upstream region of the LAT promoter may have a role in repressing LAT gene expression in cultured non-neuronal cells.

Animals↗
PubMed2006

Habitat and transmission--effect of tidal level and upstream host density on metacercarial load in an intertidal bivalve.

Transmission of parasites may be mediated by their habitat, consisting of abiotic and biotic components. I investigated the effect of 2 important habitat components in intertidal ecosystems, tidal level (abiotic) and density of upstream hosts (biotic), on the transmission of trematode cercariae to cockle (Cerastoderma edule) hosts. A field survey showed no general trend in metacercarial loads of cockles regarding tidal level but species-dependent reactions. Parasites originating from Littorina littorea (Himasthla elongata, Renicola roscovita) showed highest infection levels in the low intertidal while parasites originating from Hydrobia ulvae (H. continua, H. interrupta) showed highest infection levels in the mid-intertidal. This reflected the density of upstream hosts at both tidal levels and positive relationships between the density of upstream hosts and metacercarial load in cockles suggested the biotic habitat component to be the dominant factor in transmission. This was confirmed by a field experiment, manipulating tidal level and the density of infected upstream snail hosts. While tidal level had no significant effect on the number of metacercariae of H. elongata acquired by cockles, the effect of upstream host density was strong. In conclusion, although tidal level usually is a very important abiotic habitat component in intertidal ecosystems leading to conspicuous zonation patterns in free-living organisms, it seems of minor importance for trematode transmission. In contrast, the biotic component upstream host density is suggested to be the dominant predictor for trematode transmission to second intermediate hosts. Assessing the relative importance of abiotic and biotic habitat components in transmission is vital for the understanding of transmission processes in the field.

Animals↗
PubMed2003

Hormone-induced modifications of the chromatin structure surrounding upstream regulatory regions conserved between the mouse and rabbit whey acidic protein genes.

The upstream regulatory regions of the mouse and rabbit whey acidic protein (WAP) genes have been used extensively to target the efficient expression of foreign genes into the mammary gland of transgenic animals. Therefore both regions have been studied to elucidate fully the mechanisms controlling WAP gene expression. Three DNase I-hypersensitive sites (HSS0, HSS1 and HSS2) have been described upstream of the rabbit WAP gene in the lactating mammary gland and correspond to important regulatory regions. These sites are surrounded by variable chromatin structures during mammary-gland development. In the present study, we describe the upstream sequence of the mouse WAP gene. Analysis of genomic sequences shows that the mouse WAP gene is situated between two widely expressed genes (Cpr2 and Ramp3). We show that the hypersensitive sites found upstream of the rabbit WAP gene are also detected in the mouse WAP gene. Further, they encompass functional signal transducer and activator of transcription 5-binding sites, as has been observed in the rabbit. A new hypersensitive site (HSS3), not specific to the mammary gland, was mapped 8 kb upstream of the rabbit WAP gene. Unlike the three HSSs described above, HSS3 is also detected in the liver, but similar to HSS1, it does not depend on lactogenic hormone treatments during cell culture. The region surrounding HSS3 encompasses a potential matrix attachment region, which is also conserved upstream of the mouse WAP gene and contains a functional transcription factor Ets-1 (E26 transformation-specific-1)-binding site. Finally, we demonstrate for the first time that variations in the chromatin structure are dependent on prolactin alone.

Animals↗
PubMed2003

Upstream and intronic regulatory sequences interact in the activation of the glutamine synthetase promoter.

Glutamine synthetase (GS) is expressed at high levels in subsets of cells in some tissues and at low levels in all cells of other tissues, suggesting that the GS gene is surrounded by multiple regulatory elements. We searched for such elements in the 2.5-kb upstream region and in the 2.6-kb first intron of the GS gene, using FTO-2B hepatoma and C2/7 muscle cells as representatives of both cell types and transient transfection assays as our tools. In addition to the entire upstream region and entire intron, an upstream enhancer module at -2.5 kb, and 5', middle and 3' modules of the first intron were tested. The main effects of the respective modules and their combinatorial interactions were quantified using the analysis of variance (anova) technique. The upstream enhancer was strongly stimulatory, the middle intron module strongly inhibitory, and the 3'-intron module weakly stimulatory in both hepatoma and muscle cells. The 5'-intron module was strongly stimulatory in muscle cells only. The major new finding was that in both cell types, the upstream enhancer and 5'-intron module needed to be present simultaneously to fully realize their transactivational potencies. This interaction was responsible for a pronounced inhibitory effect of the 5'-intron module in the absence of the upstream enhancer in hepatoma cells, and for a strong synergistic effect of these two modules, when present simultaneously in muscle cells. The main difference between hepatoma and muscle cells therefore appeared to reside in tissue-specific differences in activity of the respective regulatory elements due to interactions rather than in the existence of tissue-specific regulatory elements.

Animals↗
PubMed1984

Mapping of DNase I-hypersensitive sites in the upstream DNA of human embryonic epsilon-globin gene in K562 leukemia cells.

We have mapped the DNase I-hypersensitive sites around the epsilon-globin and c-myc genes in two human leukemia cell lines K562 and HL60. In K562 cells in which the epsilon-globin gene is transcribed, six DNase I-hypersensitive sites are found in 6 kilobases (kb) of upstream flanking DNA; in HL60 cells in which the c-myc gene is expressed, two DNase I-hypersensitive sites are observed in 2 kb of upstream DNA. Neither the inactive epsilon-globin gene in HL60 cells nor the inactive c-myc gene in K562 cells displays such upstream DNase I-hypersensitive sites. Our results are consistent with previous studies that have shown DNase I-hypersensitive sites within 1 kb of the 5' end of other expressed genes. In addition, we have found sites displaying even more DNase I sensitivity further upstream of expressed epsilon-globin and c-myc genes. Among the six DNase I-hypersensitive sites of the expressed epsilon-globin gene in K562 cells, the most sensitive site is located about 6 kb upstream of the epsilon-globin gene. When correlated with the DNA sequence upstream of the epsilon-globin gene, this site was found to correspond to a region that contains a stretch of 28 consecutive Ts, three enhancer core-like sequences, and a stretch of consecutive (C-A)15(T-A)6 alternating purine and pyrimidine bases. These findings suggest the possibility that an enhancer element for epsilon-globin gene expression resides within this DNase I-hypersensitive site.

Animals↗
PubMed2006

Identification of far upstream element-binding protein-1 as an authentic Parkin substrate.

Aminoacyl-tRNA synthetase-interacting multifunctional protein type 2 was recently identified as an authentic substrate of the ubiquitin E3 ligase, parkin, a gene associated with autosomal recessive juvenile parkinsonism. Far upstream element-binding protein 1 is known to be degraded in an aminoacyl-tRNA synthetase interacting multifunctional protein type 2 dependent manner, which is crucial for lung cell maturation in early development. Therefore, we wondered whether far upstream element-binding protein 1 levels are altered in the absence of Parkin and in Parkinson disease. We herein report that far upstream element-binding protein 1 accumulates in Parkin knock-out mice, patients with autosomal recessive juvenile parkinsonism, sporadic Parkinson disease, and diffuse Lewy Body disease as well as the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson disease. Moreover, Parkin interacts with and ubiquitinates far upstream element-binding protein 1 facilitating its degradation through the ubiquitin proteasome system. Taken together, these results suggest that far upstream element-binding protein 1 is an authentic substrate of Parkin and that far upstream element-binding protein 1 might play an important role in development of Parkinson disease pathology along with aminoacyl-tRNA synthetase interacting multifunctional protein type 2.

Amino Acyl-tRNA Synthetases↗
PubMed1994

Localization of the intrinsically bent DNA region upstream of the E.coli rrnB P1 promoter.

DNA sequences upstream of the rrnB P1 core promoter (-10, -35 region) increase transcription more than 300-fold in vivo and in vitro. This stimulation results from a cis-acting DNA sequence, the UP element, which interacts directly with the alpha subunit of RNA polymerase, increasing transcription about 30-fold, and from a positively acting transcription factor, FIS, which increases expression another 10-fold. A DNA region exhibiting a high degree of intrinsic curvature has been observed upstream of the rrnB P1 core promoter and has thus been often cited as an example of the effect of bending on transcription. However, the precise position of the curvature has not been determined. We address here whether this bend is in fact related to activation of rRNA transcription. Electrophoretic analyses were used to localize the major bend in the rrnB P1 upstream region to position approximately -100 with respect to the transcription initiation site. Since most of the effect of upstream sequences on transcription results from DNA between the -35 hexamer and position -88, i.e. downstream of the bend center, these studies indicate that the curvature leading to the unusual electrophoretic behavior of the upstream region does not play a major role in activation of rRNA transcription. Minor deviations from normal electrophoretic behavior were associated with the region just upstream of the -35 hexamer and could conceivably influence interactions between the UP element and the alpha subunit of RNA polymerase.

Base Sequence↗