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PubMed1988

Identification of upstream regulatory elements involved in the developmental expression of the Arabidopsis thaliana cab1 gene.

We studied cis regulatory elements controlling the light-dependent organ-specific expression of Arabidopsis thaliana chlorophyll a/b binding protein gene (cab1) by stably transforming tobacco plants using a tumor-inducing (Ti) plasmid vector system. The results from the 5' and internal deletion analyses indicate that there are at least three cis-acting elements that are involved in the light-dependent developmental expression of cab1 gene. Two such elements are located at the immediate upstream regulatory region and the other element is located at the further upstream region. The 1120-base-pair (bp) DNA fragment containing the immediate and far upstream region can confer light-inducible organ specificity on the truncated nos promoter. However, deletion of the 39-bp DNA fragment at the immediate upstream regulatory region from this hybrid promoter resulted in a nonfunctional promoter, revealing that the 39-bp region is important for the cab promoter specificity. Further analyses of this region suggest that a potential Z-DNA-forming sequence (ATACGTGT) is involved in light-dependent developmental expression of the cab1 gene. Two additional Z-DNA-forming sequences (ACACATAT) that are inverted repeats of this sequence are also found in the upstream region where the additional regulatory elements are expected.

Base Sequence↗

PubMed1997

Multiple imprinted sense and antisense transcripts, differential methylation and tandem repeats in a putative imprinting control region upstream of mouse Igf2.

The mouse insulin-like growth factor 2 (Igf2) locus is a complex genomic region that produces multiple transcripts from alternative promoters. Expression at this locus is regulated by parental imprinting. However, despite the existence of putative imprinting control elements in the Igf2 upstream region, imprinted transcriptional repression is abolished by null mutations at the linked H19 locus. To clarify the extent to which the Igf2 upstream region contains autonomous imprinting control elements we have performed functional and comparative analyses of the region in the mouse and human. Here we report the existence of multiple, overlapping imprinted (maternally repressed) sense and antisense transcripts that are associated with a tandem repeat in the mouse Igf2 upstream region. Regions flanking the repeat exhibit tissue-specific parental allelic methylation patterns, suggesting the existence of tissue-specific control elements in the upstream region. Studies in H19 null mice indicate that both parental allelic methylation and monoallelic expression of the upstream transcripts depends on an intact H19 gene acting in cis. The homologous region in human IGF2 is structurally conserved, with the significant exception that it does not contain a tandem repeat. Our results support the proposal that tandem repeats act to target methylation to imprinted genetic loci.

Alleles↗

PubMed1998

Upstream A-tracts increase bacterial promoter activity through interactions with the RNA polymerase alpha subunit.

Upstream A-tracts stimulate transcription from a variety of bacterial promoters, and this has been widely attributed to direct effects of the intrinsic curvature of A-tract-containing DNA. In this work we report experiments that suggest a different mechanism for the effects of upstream A-tracts on transcription. The similarity of A-tract-containing sequences to the adenine- and thymine-rich upstream recognition elements (UP elements) found in some bacterial promoters suggested that A-tracts might increase promoter activity by interacting with the alpha subunit of RNA polymerase (RNAP). We found that an A-tract-containing sequence placed upstream of the Escherichia coli lac or rrnB P1 promoters stimulated transcription both in vivo and in vitro, and that this stimulation required the C-terminal (DNA-binding) domain of the RNAP alpha subunit. The A-tract sequence was protected by wild-type RNAP but not by alpha-mutant RNAPs in footprints. The effect of the A-tracts on transcription was not as great as that of the most active UP elements, consistent with the degree of similarity of the A-tract sequence to the UP element consensus. A-tracts functioned best when positioned close to the -35 hexamer rather than one helical turn farther upstream, similar to the positioning optimal for UP element function. We conclude that A-tracts function as UP elements, stimulating transcription by providing binding site(s) for the RNAP alphaCTD, and we suggest that these interactions could contribute to the previously described wrapping of promoter DNA around RNAP.

Base Sequence↗

PubMed2000

A large upstream region is not necessary for gene expression or hypersensitive site formation at the mouse beta -globin locus.

Developmental expression at the beta-globin locus is regulated in part by the locus control region, a region upstream of the genes containing at least five major DNase I hypersensitive sites (HSs) in mammalian erythrocytes. Sequences farther 5' of these HSs are conserved in mouse and human, and both loci are embedded within a cluster of functional odorant receptor genes. In humans, distant upstream sequences have been implicated in regulation of the beta-globin genes. In this study, the role of the 5'-most HSs and their adjacent sequence was investigated by deletion of an 11-kb region from the mouse locus, including 5'HS 4.2, 5'HS 5, 5'HS 6, and the 5'beta1 odorant receptor gene. Mice that were homozygous for this deletion were fully viable, and no significant effect on adult beta-globin gene expression was seen. 5'HSs 1-4, which are located downstream of the deletion, were still present in the mutant mice. In addition, two new upstream HSs, HS -60.7 and HS -62.5, were found in erythroid tissue of both wild-type and mutant mice. Therefore, although the possibility of a minor role still exists, neither the HSs nor the other regions deleted in this study are essential for beta-globin gene expression, and it is unlikely that chromatin structure is affected either upstream or downstream of the deletion. This is the largest deletion at the mouse locus control region to show no apparent phenotype, and focuses attention on the possible contribution of sequences even farther upstream.

Animals↗

PubMed2005

Coupled translation of the respiratory syncytial virus M2 open reading frames requires upstream sequences.

We have investigated the mechanism of the translation of the second open reading frame (ORF) of the respiratory syncytial virus M2 transcript that uses a novel coupled translation process requiring prior translation of the upstream ORF. The second M2-2 ORF sequences play no role in the coupling process and can be replaced with other gene sequences. Surprisingly, the overlap region of the two ORFs alone was not sufficient for coupled translation to occur. An analysis of the sequences required for the coupling process showed that portions of the transcript located along the length of the first ORF M2-1, upstream of the ORF overlap region, were essential for coupled translation to occur. A critically important region for this process was centered approximately 150 nucleotides upstream of the ORF2 initiation codons. This region was shown to contain a significant degree of secondary structure, and mutation of this sequence to remove predicted areas of base pairing significantly reduced coupled translation, confirming that the secondary structure was important for the coupling process. Additional sequences further upstream increased the efficiency of the coupled translation process. These data indicate that upstream sequences act in conjunction with the M2-1/M2-2 overlap region to promote coupled translation.

Base Sequence↗

PubMed2005

Extended upstream A-T sequence increases T7 promoter strength.

Bacteriophage T7 promoters contain a consensus sequence from -17 to +6 relative to the transcription start site, +1. In addition, the strong class III promoters are characterized by an extended AT-rich region upstream of -17, which is often interrupted by one or more GC base pairs in the weaker class II promoters. Herein we studied the role of the AT-rich region upstream of -17 in transcription regulation of T7 RNA polymerase. Equilibrium DNA binding studies with promoter fragments of consensus sequence truncated at various positions between -17 and -27 showed that the polymerase-promoter complex is significantly stabilized as the upstream AT-rich sequence is extended to and beyond -22. Similarly, promoters in which the AT-rich region from -17 to -22 is interrupted by several GC base pairs showed weak binding. Kinetic studies indicated that the presence of extended AT-rich sequence slows the dissociation rate constant of the polymerase-promoter complex and slightly stimulates the association rate constant, thereby increasing the stability of the complex. Measurement of the transcription activity revealed that the extended AT-rich region does not affect the kinetics of abortive synthesis up to the formation of 8-nucleotide RNA but causes accumulation of longer abortive products between 9 and 13 nucleotides. The observed effects of the upstream DNA region were AT sequence-specific, and the results suggested a larger role for the extended AT-rich sequence that has been unappreciated previously. We propose that the AT-rich DNA sequence upstream of -17 plays a role in modulating the efficiency of transcription initiation by affecting both the affinity of T7 RNA polymerase for the promoter and the efficiency of promoter clearance.

AT Rich Sequence↗

PubMed1986

Dioxin-inducible enhancer region upstream from the mouse P(1)450 gene and interaction with a heterologous SV40 promoter.

In mouse hepatoma Hepa-1 cells, polycyclic aromatic compounds such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) activate transcription of the mouse P(1)450 gene via trans-acting regulatory factors that include the TCDD X receptor complex. The positive control element in the P(1)450 5'-flanking region was examined in control and TCDD-treated Hepa-1 stable transformants that had been transfected with either of two expression vectors containing the chloramphenicol acetyltransferase (CAT) gene: pA10-cat, which has the simian virus 40 (SV40) early core promoter (without enhancers) immediately upstream from the CAT gene; and pSV0-cat, which has no promoter or enhancer. When the 1-kb DNA fragment from -1,647 to -611 upstream from the P(1)450 gene is inserted in either orientation--immediately upstream or almost 2 kb further upstream--from the SV40 promoter in pA10-cat, there is enhancement of CAT activity that can be further induced three- to fourfold by TCDD. When the same experiment is carried out with the -1,247 to -823 fragment or the -1,051 to -823 fragment, but not the -1,247 to -1,052 fragment, TCDD responsiveness is lost, or at least masked, because of a large increase in constitutive CAT activity. pSV0-cat mutants containing internal deletions in the upstream flanking sequences of P(1)450 were constructed. A region of 300 bases (-1,218 to -918) is shown to be required for TCDD responsiveness, and one TCDD-inducible element can be dissociated from an enhancer of constitutive gene expression, whereas one or more other TCDD-inducible elements cannot.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetyltransferases↗

PubMed2003

Upstream AUGs in embryonic proinsulin mRNA control its low translation level.

Proinsulin is expressed prior to development of the pancreas and promotes cell survival. Here we study the mechanism affecting the translation efficiency of a specific embryonic proinsulin mRNA. This transcript shares the coding region with the pancreatic form, but presents a 32 nt extended leader region. Translation of proinsulin is markedly reduced by the presence of two upstream AUGs within the 5' extension of the embryonic mRNA. This attenuation is lost when the two upstream AUGs are mutated to AAG, leading to translational efficiency similar to that of the pancreatic mRNA. The upstream AUGs are recognized as initiator codons, because expression of upstream ORF is detectable from the embryonic transcript, but not from the mutated or the pancreatic mRNAs. Strict regulation of proinsulin biosynthesis appears to be necessary, since exogenous proinsulin added to embryos in ovo decreased apoptosis and generated abnormal developmental traits. A novel mechanism for low level proinsulin expression thus relies on upstream AUGs within a specific form of embryonic proinsulin mRNA, emphasizing its importance as a tightly regulated developmental signal.

3T3 Cells↗

PubMed2001

Effect of allelic variation at the NACP-Rep1 repeat upstream of the alpha-synuclein gene (SNCA) on transcription in a cell culture luciferase reporter system.

Mutations in the alpha-synuclein gene (SNCA) have been implicated in familial Parkinson's disease (PD) while certain polymorphic alleles at a microsatellite repeat, NACP-Rep1, located approximately 10 kb upstream of the gene, have been associated with sporadic PD. In order to study the regulation of the human alpha-synuclein gene, we performed a deletion analysis of 10.7 kb upstream of the translational start site, using the luciferase reporter assay in 293T cells and the neuroblastoma cell line SH-SY5Y. The shortest fragment, 400 bp upstream of the transcriptional start site, was sufficient for transcription in both cell lines. The other constructs led to variable expression levels, with some showing maximum expression and others showing nearly complete extinction of expression. An 880 bp fragment located approximately 10 kb upstream of the gene and containing the NACP-Rep1 polymorphism, was shown to be necessary for normal expression. Additional analysis of the NACP-Rep1 locus and surrounding DNA suggested that two domains flanking the repeat interact to enhance expression while the repeat acts as a negative modulator. Next, we measured the activity of the entire 10.7 kb upstream region in the luciferase reporter assay when each of our different NACP-Rep1 alleles were present. The expression levels varied very significantly among the different alleles over a 3-fold range in the SH-SY5Y cells but showed little or no significant variation in the 293T cells. Given that even small changes in alpha-synuclein expression may, over many decades, predispose to PD, the association of different NACP-Rep1 alleles with PD may be a consequence of polymorphic differences in transcriptional regulation of alpha-synuclein expression resulting from different NACP-Rep1 alleles.

Alleles↗

PubMed1984

Alternative sites of transcription initiation upstream of the canonical cap site in human gamma-globin and beta-globin genes.

Using S1 mapping and primer extension analysis, we have identified a number of human kappa-globin and beta-globin 5' RNA termini originating in the 200 bp upstream of the canonical mRNA cap sites. Upstream initiation sites have previously been reported for the human epsilon-globin gene (4,5) and the present work indicates that this is a general feature of the human beta-type globin genes. We have attempted to identify features common to such sites between the three genes. One site 170 bp upstream of the major beta-globin cap site and a site 1400 bp upstream of the major epsilon-globin cap site are located near putative PolIII promoter sequences and may therefore be transcribed by this enzyme. Alternative initiation sites located 200 bp and 50-100 bp upstream of the epsilon-globin and kappa-globin cap sites respectively are located within S1 hypersensitive regions of chromatin.

Base Composition↗

PubMed1985

Characterization of an unique RNA initiated immediately upstream from human alpha 1 globin gene in vivo and in vitro: polymerase II-dependence, tissue specificity, and subcellular location.

We have identified an abundant transcript initiated upstream from the canonical cap site of human alpha 1 globin gene in bone marrow cells and in COS-7 cells transfected with an alpha 1 globin gene-containing plasmid. Similar to the major alpha 1 globin transcript, this upstream RNA is present almost exclusively in the cytoplasm of the transfected COS-7 cells. It is also synthesized efficiently in vitro by RNA polymerase II in the nuclear extracts prepared from a Hela cell line and an erythroleukemia cell line, K562. RNAs isolated from these cell lines, however, do not contain this upstream transcript. The putative 5' end of the alpha 1 globin upstream RNA is mapped by primer extension to base -45, which is located in between the CCAAT and TATA boxes. The synthesis of this RNA in vitro and in vivo, and the close proximity of its 5' end to the promoter of the alpha 1 globin gene suggest a common mechanism regulating the transcriptional initiation of both the upstream and the major alpha 1 globin RNAs.

Animals↗

PubMed1988

Nucleotide sequence of the LuxC gene and the upstream DNA from the bioluminescent system of Vibrio harveyi.

The nucleotide sequence of the luxC gene (1431 bp) and the upstream DNA (1049 bp) of the luminescent bacterium Vibrio harveyi has been determined. The luxC gene can be translated into a polypeptide of 55 kDa in excellent agreement with the molecular mass of the reductase polypeptide required for synthesis of the aldehyde substrate for the bioluminescent reaction. Analyses of codon usage showed a high frequency (1.9%) of the isoleucine codon, AUA, in the luxC gene compared to that found in Escherichia coli genes (0.2%) and its absence in the luxA, B and D genes. The low G/C content of the luxC gene and upstream DNA (38-39%) compared to that found in the other lux genes of V. harveyi (45%) was primarily due to a stretch of 500 nucleotides with only a 24% G/C content, extending from 200 bp inside lux C to 300 bp upstream. Moreover, an open reading frame did not extend for more than 48 codons between the luxC gene and 600 bp upstream at which point a gene transcribed in the opposite direction started. As the lux system in the luminescent bacterium, V. fischeri, contains a regulatory gene immediately upstream of luxC transcribed in the same direction, these results show that the organization and regulation of the lux genes have diverged in different luminescent bacteria.

Amino Acid Sequence↗

PubMed1989

Molecular analysis by deletion and site-directed mutagenesis of the cis-acting upstream sequence involved in activation of the ompF promoter in Escherichia coli.

Expression of the ompF gene coding for an outer membrane protein of Escherichia coli is regulated by a transcriptional activation mechanism that requires the ompR gene product that acts on nucleotides located upstream of the -35 and -10 regions of the ompF promoter. We previously demonstrated that this cis-acting upstream sequence displays a sequence-directed curvature of the DNA helix. To characterize the structure and function of this upstream sequence, a series of deletion mutants and base-substitution mutants of the upstream sequence of the ompF promoter were constructed, and their abilities as to OmpR-binding and activities of the ompF promoter were examined after they had been connected to the lacZ gene. The nucleotides extending from position -91 to -79 are essential not only for sequence-specific recognition of the ompF promoter by the OmpR protein, but also for OmpR-dependent activation of the ompF promoter. It was also demonstrated that the nucleotides extending from position -111 to -92 play a role in stimulation of the ompF expression. A local structural alteration in the ompF promoter was observed in some of the base-substitution mutants. Based on the results, the structure and function of the upstream sequence of the ompF promoter are discussed in relation to activation of the ompF promoter by the OmpR protein.

Bacterial Outer Membrane Proteins↗

PubMed1996

The role of TBP in rDNA transcription by RNA polymerase I in Saccharomyces cerevisiae: TBP is required for upstream activation factor-dependent recruitment of core factor.

Transcription of Saccharomyces cerevisiae rDNA by RNA polymerase I involves at least two transcription factors characterized previously: upstream activation factor (UAF) consisting of Rrn5p, Rrn9p, Rrn10p, and two more uncharacterized proteins; and core factor (CF) consisting of Rrn6p, Rrn7p, and Rrn11p. UAF interacts directly with an upstream element of the promoter and mediates its stimulatory function, and CF subsequently joins a stable preinitiation complex. The TATA-binding protein (TBP) has been known to be involved in transcription by all three nuclear RNA polymerases. We found that TBP interacts specifically with both UAF and CF, the interaction with UAF being stronger than that with CF. Using extracts from a TBP (I143N) mutant, it was shown that TBP is required for stimulation of transcription mediated by the upstream element, but not for basal transcription directed by a template without the upstream element. By template competition experiments, it was shown that TBP is required for UAF-dependent recruitment of CF to the rDNA promoter, explaining the TBP requirement for stimulatory activity of the upstream element. We also studied protein-protein interactions and found specific interactions of TBP with Rrn6p and with Rrn9p both in vitro and in the yeast two-hybrid system in vivo. Thus, these two interactions may be involved in the interactions of TBP with CF and UAF, respectively, contributing to the recruitment of CF to the rDNA promoter. Additionally, we observed an interaction between Rrn9p and Rrn7p both in vitro and in the two-hybrid system; thus, this interaction might also contribute to the recruitment of CF.

DNA, Ribosomal↗

PubMed1988

Specific binding of proteins from Rhizobium meliloti cell-free extracts containing NodD to DNA sequences upstream of inducible nodulation genes.

Nodulation (nod) genes in Rhizobium meliloti are transcriptionally induced by flavonoid signal molecules, such as luteolin, produced by its symbiotic host plant, alfalfa. This induction depends on expression of nodD. Upstream of three inducible nod gene clusters, nodABC, nodFE, and nodH, is a highly conserved sequence referred to as a 'nod box.' The upstream sequences have no other obvious similarity. We have found that DNA fragments containing the regions upstream of all three inducible transcripts show altered electrophoretic mobility when treated with R. meliloti extracts. The ability of the extracts to interact specifically with these DNAs correlated with the genetic dosage of nodD1 or nodD3 and with the presence and concentration of the nodD1 or nodD3 protein (NodD1 or NodD3) in the extracts. Antiserum specific to NodD was used to construct an immunoaffinity column that permitted a substantial purification of NodD1; this preparation of NodD1 also displayed specific binding to restriction fragments containing DNA sequences found upstream of inducible nod genes. In addition, NodD-specific antiserum removed the specific DNA-binding activity from total Rhizobium cell extracts. The interaction of total extracts and of partially purified NodD protein with nod promoter sequences was competitive with an oligonucleotide representing the 3' 25-bp portion of the nod box. The interaction of R. meliloti extracts and NodD1 protein with nod gene upstream regions occurred independently of exposure of cells or extracts to flavone inducer.

Bacterial Proteins↗

PubMed2004

The human L1 promoter: variable transcription initiation sites and a major impact of upstream flanking sequence on promoter activity.

Human L1 elements are non-LTR retrotransposons that comprise approximately 17% of the human genome. Their 5'-untranslated region (5'-UTR) serves as a promoter for L1 transcription. Now we find that transcription initiation sites are not restricted to nucleotide +1 but vary considerably in both downstream and upstream directions. Transcription initiating upstream explains additional nucleotides often seen between the 5'-target site duplication and the L1 start site. A higher frequency of G nucleotides observed upstream from the L1 can be explained by reverse transcription of the L1 RNA 5'-CAP, which is further supported by extra Gs seen for full-length HERV-W pseudogenes. We assayed 5'-UTR promoter activities for several full-length human L1 elements, and found that upstream flanking cellular sequences strongly influence the L1 5'-UTR promoter. These sequences either repress or enhance the L1 promoter activity. Therefore, the evolutionary success of a human L1 in producing progeny depends not only on the L1 itself, but also on its genomic integration site. The promoter mechanism of L1 is reminiscent of initiator (Inr) elements that are TATA-less promoters expressing several cellular genes. We suggest that the L1 5'-UTR is able to form an Inr element that reaches into upstream flanking sequence.

5' Untranslated Regions↗

PubMed1990

Upstream sequences other than AAUAAA are required for efficient messenger RNA 3'-end formation in plants.

We have characterized the upstream nucleotide sequences involved in mRNA 3'-end formation in the 3' regions of the cauliflower mosaic virus (CaMV) 19S/35S transcription unit and a pea gene encoding ribulose-1,5-bisphosphate carboxylase small subunit (rbcS). Sequences between 57 bases and 181 bases upstream from the CaMV polyadenylation site were required for efficient polyadenylation at this site. In addition, an AAUAAA sequence located 13 bases to 18 bases upstream from this site was also important for efficient mRNA 3'-end formation. An element located between 60 bases and 137 bases upstream from the poly(A) addition sites in a pea rbcS gene was needed for functioning of these sites. The CaMV -181/-57 and rbcS -137/-60 elements were different in location and sequence composition from upstream sequences needed for polyadenylation in mammalian genes, but resembled the signals that direct mRNA 3'-end formation in yeast. However, the role of the AAUAAA motif in 3'-end formation in the CaMV 3' region was reminiscent of mRNA polyadenylation in animals. We suggest that multiple elements are involved in mRNA 3'-end formation in plants, and that interactions of different components of the plant polyadenylation apparatus with their respective sequence elements and with each other are needed for efficient mRNA 3'-end formation.

Base Sequence↗

PubMed1993

Translational initiation at the coat-protein gene of phage MS2: native upstream RNA relieves inhibition by local secondary structure.

Maximal translation of the coat-protein gene from RNA bacteriophage MS2 requires a contiguous stretch of native MS2 RNA that extends hundreds of nucleotides upstream from the translational start site. Deletion of these upstream sequences from MS2 cDNA plasmids results in a 30-fold reduction of translational efficiency. By site-directed mutagenesis, we show that this low level of expression is caused by a hairpin structure centred around the initiation codon. When this hairpin is destabilized by the introduction of mismatches, expression from the truncated messenger increases 20-fold to almost the level of the full-length construct. Thus, the translational effect of hundreds of upstream nucleotides can be mimicked by a single substitution that destabilizes the structure. The same hairpin is also present in full-length MS2 RNA, but there it does not impair ribosome binding. Apparently, the upstream RNA somehow reduces the inhibitory effect of the structure on translational initiation. The upstream MS2 sequence does not stimulate translation when cloned in front of another gene, nor can unrelated RNA segments activate the coat-protein gene. Several possible mechanisms for the activation are discussed and a function in gene regulation of the phage is suggested.

Base Sequence↗