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At least 289 records · Page 16Linked to original sources
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↗
PubMed2000

The VirR response regulator from Clostridium perfringens binds independently to two imperfect direct repeats located upstream of the pfoA promoter.

Regulation of toxin production in the gram-positive anaerobe Clostridium perfringens occurs at the level of transcription and involves a two-component signal transduction system. The sensor histidine kinase is encoded by the virS gene, while its cognate response regulator is encoded by the virR gene. We have constructed a VirR expression plasmid in Escherichia coli and purified the resultant His-tagged VirR protein. Gel mobility shift assays demonstrated that VirR binds to the region upstream of the pfoA gene, which encodes perfringolysin O, but not to regions located upstream of the VirR-regulated plc, colA, and pfoR genes, which encode alpha-toxin, collagenase, and a putative pfoA regulator, respectively. The VirR binding site was shown by DNase I footprinting to be a 52-bp core sequence situated immediately upstream of the pfoA promoter. When this region was deleted, VirR was no longer able to bind to the pfoA promoter. The binding site was further localized to two imperfect direct repeats (CCCAGTTNTNCAC) by site-directed mutagenesis. Binding and protection analysis of these mutants indicated that VirR had the ability to bind independently to the two repeated sequences. Based on these observations it is postulated that the VirR positively regulates the synthesis of perfringolysin O by binding directly to a region located immediately upstream of the pfoA promoter and activating transcription.

Bacterial Proteins↗
PubMed1988

Translational control of cytomegalovirus gene expression is mediated by upstream AUG codons.

Human cytomegalovirus regulates gene expression at both the transcriptional and posttranscriptional levels. We have characterized posttranscriptional signals carried on the RNA of one beta (or delayed-early) gene, signals that we show delay the translation of an actively transcribed gene. The cis-acting components of this signal map to two upstream AUG codons in the 5' leader of the beta gene transcript that act to delay the kinetics and reduce the levels of gene expression. Both upstream AUG codons are part of short open reading frames, and the presence of either is sufficient to suppress expression of a downstream open reading frame. These upstream natural AUG codons appear to act in cis at the level of translation, consistent with the scanning hypothesis of eucaryotic translation. The occurrence of natural upstream AUG codons in other cytomegalovirus transcripts suggests their physiological significance in influencing the kinetics and steady-state levels of gene expression during viral growth.

Cells, Cultured↗
PubMed1992

A constitutive enhancer in the bovine papillomavirus upstream regulatory region shares genetic elements with the viral P1 promoter.

The bovine papillomavirus upstream regulatory region represents a common element in the regulation of transcription from the five early viral promoters. We have determined the sequences required for transcription from the viral P1 promoter, which is located at the 5' end of the upstream regulatory region. In vitro transcription from P1 requires a 123-bp fragment (nucleotides 7153 to 7275; -33 to +90) consisting of an upstream TATA-like sequence as well as an unidentified protein which binds to sequences immediately downstream of the initiation site. In vivo, this promoter requires additional downstream sequences (to position +160; nucleotide 7345) for maximal activity but does not require any additional DNA sequence upstream of a putative TATA box. Four regions within the downstream sequence from +9 to +160 are protected from DNase I digestion by proteins present in a HeLa cell extract. The presence of these sites correlates with the level of P1 activity. A constitutive enhancer maps to this same region, and mutations in this enhancer have been shown to affect downstream promoters. Deletion analysis indicates that the same sequences are required by both the P1 promoter and the constitutive enhancer, suggesting that the same proteins function in both activities.

Animals↗
PubMed1992

Upstream sequences and cap proximity in the regulation of polyadenylation in ground squirrel hepatitis virus.

The polyadenylation signal of mammalian hepadnaviruses is unusual in that its hexanucleotide element is the variant UAUAAA rather than AAUAAA. This signal functions inefficiently and must be augmented by multiple activator elements located in the upstream 400 nucleotides (nt) to promote efficient processing. Here we characterize one of these upstream elements, termed PS2, in the ground squirrel hepatitis virus. PS2 is located within the 107 nt 5' to the UAUAAA and raises the efficiency of polyadenylation by this signal from < 10% to 50 to 60%. It can function independently of the more 5' activator elements and conversely is not required for their function. Its action is orientation dependent, and a predicted stem-loop structure within the element is not necessary for its activity. PS2 is the sole upstream element that maps within the terminal redundancy of viral genomic RNA. Thus, it is present, together with the UAUAAA, at both the 5' and 3' ends of this RNA. During genomic RNA synthesis, the poly(A) signals in the 5' repeat are bypassed, while those in the 3' copy are used. The ability of PS2 to function independently of the other, more upstream activators suggests that the absence of the latter elements from the 5' redundancy is insufficient to account for bypass of the 5' poly(A) site, as we had earlier proposed. Rather, the short distance from the cap site to the UAUAAA at the 5' end of genomic RNA actively suppresses its use, as this suppression can be experimentally relieved by increasing this distance to 230 to 400 nt.

Animals↗
PubMed1992

Two distant upstream regions containing cis-acting signals regulating splicing facilitate 3'-end processing of avian sarcoma virus RNA.

Retroviruses, pararetroviruses, and related retrotransposons generate terminally redundant RNAs by transcription of a template flanked by long terminal repeats in which initiation occurs within the 5' long terminal repeat sequences and 3'-end processing occurs within the 3' long terminal repeat sequences. Processing of avian sarcoma virus RNA is relatively inefficient; approximately 15% of the viral RNA transcripts are read-through products; i.e., they are not processed at the viral poly(A) addition site but at sites in the cellular sequence further downstream. In this report, we show that the efficiency of processing at the viral site is further reduced by deletion of two distant upstream sequences: (i) a 606-nucleotide sequence in the gag gene containing a cis-acting negative regulator of splicing and (ii) a 136-nucleotide sequence spanning the env 3' splice site. The deletion of either or both upstream regions increases the levels of read-through products of both unspliced and spliced viral RNA. In contrast, deletion of the src 3' splice site does not affect the efficiency of processing at the viral poly(A) addition site. The effects on 3'-end processing are not correlated either with distance from the promoter to the poly(A) addition site or with the overall level of viral RNA splicing. Substitution of the avian sarcoma virus poly(A) signal with the simian virus 40 early or late poly(A) signal relieves the requirement for the distant upstream sequences. We propose that cellular factors, which may correspond to splicing factors, bound to the upstream viral sequences may interact with factors bound at the avian sarcoma virus poly(A) signal to stabilize the polyadenylation-cleavage complex and allow for more efficient 3'-end processing.

Animals↗
PubMed1995

Efficient excision of the upstream large intron from P4-generated pre-mRNA of the parvovirus minute virus of mice requires at least one donor and the 3' splice site of the small downstream intron.

We have previously shown that efficient excision of the upstream large intron from P4-generated pre-mRNA of the autonomous parvovirus minute virus of mice depends upon at least the initial presence of sequences within the downstream small intron (Q. Zhao, R. V. Schoborg, and D. J. Pintel, J. Virol. 68:2849-2859, 1994). In this report, we show that the requirement of downstream small intron sequences is complex and that efficient excision of the upstream intron requires at least one small intron donor and the 3' splice site. In the absence of both small intron donors, a new spliced product is produced in which the intervening exon is skipped and the large intron donor at nucleotide 514 is joined to a small intron acceptor. Exon skipping caused by the loss of the two small intron donors can be overcome, and the excision of the large intron can be regained by mutations that improve the large intron polypyrimidine tract. These results are consistent with a model in which the binding of multiple splicing factors that assemble at both a downstream donor and acceptor facilitates the binding of splicing factors to the weak polypyrimidine tract of the upstream large intron, thereby defining the intervening exon and promoting excision of the upstream intron.

Animals↗
PubMed2004

Alternative polyadenylation of adeno-associated virus type 5 RNA within an internal intron is governed by both a downstream element within the intron 3' splice acceptor and an element upstream of the P41 initiation site.

Adeno-associated virus type 5 (AAV5) has a linear, single-stranded DNA genome of ca. 5 kb and an overlapping transcription profile featuring multiple promoters and a single intron in the center of the genome. Unlike the situation for the prototype AAV2, AAV5 RNAs transcribed from upstream promoters at map units 7 (P7) and 19 (P19), which encode the viral Rep proteins, are predominantly polyadenylated at a site within the intron [(pA)p]. RNAs generated from the AAV5 capsid gene promoter P41, which is only 78 nucleotides (nt) upstream of the intron donor, and 281 nt upstream of (pA)p, primarily readthrough (pA)p, are polyadenylated at a more distal site at the 3' end of the genome [(pA)d] and ultimately spliced. The intron contains the core sequences sufficient for polyadenylation at (pA)p, which is governed by a G/U-rich downstream element that overlaps with the intron 3' A2 splice acceptor. In addition, polyadenylation of P7- and P19-generated RNAs at (pA)p is influenced by an upstream element that lies 5' to the start of the P41 transcript. Our results also suggest that splicing and polyadenylation of P41-generated RNA can compete for the same pool of precursor pre-mRNA molecules. The cis-acting signals within the A2 3' splice site that govern polyadenylation and splicing of AAV5 RNAs must be optimized to program both (i) the levels of polyadenylation of P7- and P19-generated RNA at (pA)p required to generate the proper levels of the essential Rep proteins and (ii) the splicing of P41-generated RNAs to generate the proper ratio of capsid proteins during AAV5 infection.

3' Untranslated Regions↗
PubMed1990

Upstream activation and repression elements control transcription of the yeast COX5b gene.

The Saccharomyces cerevisiae COX5b gene is regulated at the level of transcription by both the carbon source and oxygen. To define the cis-acting elements that underlie this transcriptional control, deletion analysis of the upstream regulatory region of COX5b was performed. The results of the study suggest that at least four distinct regulatory sites are functional upstream of the COX5b transcriptional starts. One, which was precisely defined to a region of 20 base pairs, contains two TATA-like elements. Two upstream activating sequences (UAS15b and UAS2(5b)) and an upstream repression sequence (URS5b) were also found. Each of the latter three elements was able either to activate (UAS1(5b) and UAS2(5b)) or to repress URS5b) the transcription of a heterologous yeast gene. Further analysis revealed that UAS1(5b) is the site of carbon source control and may be composed of two distinct domains that act synergistically. URS5b mediates the aerobic repression of COX5b and contains two sequences that are highly conserved in other yeast genes negatively regulated by oxygen.

Base Sequence↗
PubMed1992

The core promoter region of the tumor necrosis factor alpha gene confers phorbol ester responsiveness to upstream transcriptional activators.

Activators of protein kinase C, such as 12-O-tetradecanoylphorbol 13-acetate (TPA), are known to regulate the expression of many genes, including the tumor necrosis factor alpha (TNF) gene, by affecting the level or activity of upstream transcription factors. To investigate the mechanism whereby TPA activates the TNF promoter, a series of 5'-deletion mutants of the human TNF promoter linked to chloramphenicol acetyltransferase was transfected into U937 human promonocytic cells. TPA produced a 7- to 11-fold activation of all TNF promoters tested, even those promoters truncated to contain only the core promoter with no upstream enhancer elements. The proximal TNF promoter containing only 28 nucleotides upstream and 10 nucleotides downstream of the RNA start site confers TPA activation to a variety of unrelated upstream enhancer elements and transcription factors, including Sp1, CTF/NF1, cyclic AMP-response element, GAL-E1a, and GAL-VP16. The level of activation by TPA depends on the TATA box structure, since the TPA response is greater in promoters containing the sequence TATAAA than in those containing TATTAA or TATTTA. These findings suggest that the core promoter region is a target for gene regulation by second-messenger pathways.

Base Sequence↗
PubMed2000

Negative and translation termination-dependent positive control of FLI-1 protein synthesis by conserved overlapping 5' upstream open reading frames in Fli-1 mRNA.

The proto-oncogene Fli-1 encodes a transcription factor of the ets family whose overexpression is associated with multiple virally induced leukemias in mouse, inhibits murine and avian erythroid cell differentiation, and induces drastic perturbations of early development in Xenopus. This study demonstrates the surprisingly sophisticated regulation of Fli-1 mRNA translation. We establish that two FLI-1 protein isoforms (of 51 and 48 kDa) detected by Western blotting in vivo are synthesized by alternative translation initiation through the use of two highly conserved in-frame initiation codons, AUG +1 and AUG +100. Furthermore, we show that the synthesis of these two FLI-1 isoforms is regulated by two short overlapping 5' upstream open reading frames (uORF) beginning at two highly conserved upstream initiation codons, AUG -41 and GUG -37, and terminating at two highly conserved stop codons, UGA +35 and UAA +15. The mutational analysis of these two 5' uORF revealed that each of them negatively regulates FLI-1 protein synthesis by precluding cap-dependent scanning to the 48- and 51-kDa AUG codons. Simultaneously, the translation termination of the two 5' uORF appears to enhance 48-kDa protein synthesis, by allowing downstream reinitiation at the 48-kDa AUG codon, and 51-kDa protein synthesis, by allowing scanning ribosomes to pile up and consequently allowing upstream initiation at the 51-kDa AUG codon. To our knowledge, this is the first example of a cellular mRNA displaying overlapping 5' uORF whose translation termination appears to be involved in the positive control of translation initiation at both downstream and upstream initiation codons.

3T3 Cells↗
PubMed2005

Potential autoregulation of transcription factor PU.1 by an upstream regulatory element.

Regulation of the hematopoietic transcription factor PU.1 (Spi-1) plays a critical role in the development of white cells, and abnormal expression of PU.1 can lead to leukemia. We previously reported that the PU.1 promoter cannot induce expression of a reporter gene in vivo, and cell-type-specific expression of PU.1 in stable lines was conferred by a 3.4-kb DNA fragment including a DNase I hypersensitive site located 14 kb upstream of the transcription start site. Here we demonstrate that this kb -14 site confers lineage-specific reporter gene expression in vivo. This kb -14 upstream regulatory element contains two 300-bp regions which are highly conserved in five mammalian species. In Friend virus-induced erythroleukemia, the spleen focus-forming virus integrates into the PU.1 locus between these two conserved regions. DNA binding experiments demonstrated that PU.1 itself and Elf-1 bind to a highly conserved site within the proximal homologous region in vivo. A mutation of this site abolishing binding of PU.1 and Elf-1 led to a marked decrease in the ability of this upstream element to direct activity of reporter gene in myelomonocytic cell lines. These data suggest that a potential positive autoregulatory loop mediated through an upstream regulatory element is essential for proper PU.1 gene expression.

Animals↗
PubMed1984

Upstream region required for regulated expression of the glucose-repressible SUC2 gene of Saccharomyces cerevisiae.

The SUC2 gene produces two mRNAs with different 5' ends that encode two forms of invertase. The 1.9-kilobase mRNA encoding secreted invertase is regulated by glucose repression (carbon catabolite repression), and the 1.8-kilobase mRNA encoding intracellular invertase is produced constitutively at low levels. To identify 5' noncoding sequences essential for regulated expression of SUC2, we constructed in vitro a series of deletions and inserted them into the yeast genome at the chromosomal SUC2 locus. Analysis of the effects of each deletion on SUC2 gene expression identified an upstream region required for derepression of secreted invertase synthesis. The 3' boundary of this region is near -418. The 5' boundary does not appear to be sharply defined, but lies ca. 100 base pairs upstream. A deletion extending from -418 to -140 allowed high-level derepression, indicating that no essential sequences lie between the upstream region and the TATA box at -133 and that the upstream region can be moved 279 base pairs closer to the transcriptional start site. Interactions between the deletions and several unlinked mutations affecting the regulation of SUC2 gene expression were examined. Sequences between -1,900 and -86 are dispensable for expression of the 1.8-kilobase mRNA.

Base Sequence↗
PubMed1986

Duplicate upstream activating sequences in the promoter region of the Saccharomyces cerevisiae GAL7 gene.

We constructed a series of deletions in the 5' noncoding region of the Saccharomyces cerevisiae GAL7 gene, fused them to the Escherichia coli gene lacZ, and introduced them into yeasts by using a multicopy vector. We then studied the effect of the deletions on beta-galactosidase synthesis directed by the gene fusions in media with various carbon sources. This analysis identified a TATA box and two upstream activating sequences as necessary elements for galactose-controlled GAL7 transcription. Two upstream activating sequences exhibiting 71% homology with each other were located 255 and 168 base pairs, respectively, upstream of the GAL7 transcription start point. Each sequence consists of 21 base pairs, displaying an approximate rotational symmetry with a core consensus sequence of GAA--AGCTGCTTC--CGCG. At least one of the two sequences is required for galactose induction and also for glucose repression of the GAL7'-lac'Z gene. Analysis with host regulatory mutants delta gal14 and delta gal180 suggests that these sequences are the site at which the GAL4 product exerts its action to activate the GAL7 gene. We also observed that a deletion lacking both upstream activation sequences allowed the gene fusion to be expressed in the absence of galactose at about 10% of the fully induced level of the intact fusion. This constitutive expression depended on the presence of the TATA box of GAL7 in cis but not on a functional GAL4 gene. The level of the uncontrolled expression was decreased by increasing the distance between the TATA box and the pBR322 sequence in the vector plasmid.

Base Sequence↗
PubMed1986

Precise assignment of the light-strand promoter of mouse mitochondrial DNA: a functional promoter consists of multiple upstream domains.

Using deletion mutagenesis we localized the promoter for the light strand of mouse mitochondrial DNA to a 97-base-pair region, from -88 to +9 nucleotides of the transcriptional initiation site. Within this region the light-strand promoter could be dissected into at least three different functional domains. The specificity region, a maximum of 19 base pairs between -10 and +9 of the transcriptional initiation site, was essential and sufficient for accurate transcriptional initiation. A second region, extending to -29 nucleotides from the initiation site, facilitated the formation of a preinitiation complex between the template DNA and factor(s) present in the mitochondrial RNA polymerase fraction and was required for efficient transcription. A third, ill-defined upstream region, which extended up to -88 nucleotides from the initiation site, appeared to influence template transcriptional efficiencies in competition assays. Without the specificity domain, the upstream regions were incapable of supporting any transcription. The presence of multiple upstream domains was confirmed by disrupting nucleotide sequences in the upstream region by using linker insertion and linker replacement techniques.

Animals↗
PubMed1987

Upstream G gamma-globin and downstream beta-globin sequences required for stage-specific expression in transgenic mice.

The human G gamma-globin and beta-globin genes are expressed in erythroid cells at different stages of human development, and previous studies have shown that the two cloned genes are also expressed in a differential stage-specific manner in transgenic mice. The G gamma-globin gene is expressed only in murine embryonic erythroid cells, while the beta-globin gene is active only at the fetal and adult stages. In this study, we analyzed transgenic mice carrying a series of hybrid genes in which different upstream, intragenic, or downstream sequences were contributed by the beta-globin or G gamma-globin gene. We found that hybrid 5'G gamma/3'beta globin genes containing G gamma-globin sequences upstream from the initiation codon were expressed in embryonic erythroid cells at levels similar to those of an intact G gamma-globin transgene. In contrast, beta-globin upstream sequences were insufficient for expression of 5'beta/3'G gamma hybrid globin genes or a beta-globin-metallothionein fusion gene in adult erythroid cells. However, beta-globin downstream sequences, including 212 base pairs of exon III and 1,900 base pairs of 3'-flanking DNA, were able to activate a 5'G gamma/3'beta hybrid globin gene in fetal and adult erythroid cells. These experiments suggest that positive regulatory elements upstream from the G gamma-globin and downstream from the beta-globin gene are involved in the differential expression of the two genes during development.

Animals↗
PubMed1989

Analysis of pre-rRNAs in heat-shocked HeLa cells allows identification of the upstream termination site of human polymerase I transcription.

Human rRNA precursor from normal or stressed HeLa cells were studied by S1 nuclease mapping of unlabeled RNA and by antisense RNase mapping of RNA from cells that had been labeled in vivo with [32P]PO4. Heating cells to 43 degrees C decreased the amount of newly synthesized rRNA to less than 5% of the control level and led to greater than 95% inhibition of transcription termination at a region 355 to 362 nucleotides downstream of the 3' end of 28S rRNA, with readthrough continuing into the next transcription unit. Heating of cells to 42 degrees C led to 60% inhibition of termination at this site; 50% of transcripts that extended into the nontranscribed spacer ended in a region 200 to 210 nucleotides upstream of the polymerase I (Pol I) initiation site. This is presumed to be the human upstream transcription termination site because of the absence of RNAs with a 5' end corresponding to this region, the location relative to the Pol I initiation site (which is similar to the location of upstream terminators in other species), and the fact that it is 15 to 25 nucleotides upstream of the sequence GGGTTGACC, which has an 8-of-9 base identity with the sequence 3' of the downstream termination site. Surprisingly, treatment of cells with sodium arsenite, which also leads to the induction of a stress response, did not inhibit termination. Pol I initiation was decreased to the same extent as termination, which lends support to the hypothesis that termination and initiation are coupled. Although termination was almost completely inhibited at 43 degrees C, the majority of the recently synthesized rRNAs were processed to have the correct 3' end of 28S. This finding suggests that 3'-end formation can involve an endonucleolytic cut and is not solely dependent on exonucleolytic trimming of correctly terminated rRNAs.

Animals↗
PubMed1987

Upstream pressure for systemic to pulmonary flow from bronchial circulation in dogs.

Systemic to pulmonary flow from bronchial circulation, important in perfusing potentially ischemic regions distal to pulmonary vascular obstructions, depends on driving pressure between an upstream site in intrathoracic systemic arterial network and pulmonary vascular bed. The reported increase of pulmonary infarctions in heart failure may be due to a reduction of this driving pressure. We measured upstream element for driving pressure for systemic to pulmonary flow from bronchial circulation by raising pulmonary venous pressure (Ppv) until the systemic to pulmonary flow from bronchial circulation ceased. We assumed that this was the same as upstream pressure when there was flow. Systemic to pulmonary flow from bronchial circulation was measured in left lower lobes (LLL) of 21 anesthetized open-chest dogs from volume of blood that overflowed from pump-perfused (90-110 ml/min) pulmonary vascular circuit of LLL and was corrected by any changes of LLL fluid volume (wt). Systemic to pulmonary flow from bronchial circulation upstream pressure was linearly related to systemic arterial pressure (slope = 0.24, R = 0.845). Increasing Ppv caused a progressive reduction of systemic to pulmonary flow from bronchial circulation, which stopped when Ppv was 44 +/- 6 cmH2O and pulmonary arterial pressure was 46 +/- 7 cmH2O. A further increase in Ppv reversed systemic to pulmonary flow from bronchial circulation with blood flowing back into the dog. When net systemic to pulmonary flow from bronchial circulation by the overflow and weight change technique was zero a small bidirectional flow (3.7 +/- 2.9 ml.min-1 X 100 g dry lobe wt-1) was detected by dispersion of tagged red blood cells that had been injected.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗