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At least 73 records · Page 4Linked to original sources
PubMed1991

Contributory effects of de novo transcription and premature transcript termination in the regulation of human epidermal growth factor receptor proto-oncogene RNA synthesis.

Overexpression of the epidermal growth factor (EGF) receptor (c-erbB) proto-oncogene is a frequent occurrence in human carcinoma and appears to accompany autocrine or paracrine transforming growth factor-alpha expression, which in model systems can result in activation of EGF receptor tyrosine kinase activity and phenotypic transformation. Here we have investigated the transcriptional regulation of the EGF receptor gene, by run-on transcription in isolated nuclei derived from epithelioid tumor lines. The level of transcription was measured at various points on the 100-kilobase pair EGF receptor gene locus, on either sense or antisense DNA strands. We find the level of sense strand transcription along exon 1 is 8-fold higher than transcription in exons 2-26. Primary EGF receptor transcripts appear to pause or terminate prematurely between exons 1 and 2. Termination was mapped to a sequenced region approximately 2 kilobase pairs 3' of exon 1, proximal to a previously reported DNase I hypersensitive site and an enhancer-like activity. Transcription in the CpG-rich region surrounding exon 1 is bidirectional, with antisense transcripts initiating in intron 1 and extending through the coding first exon. Activation of protein kinase C results in a 5-fold induction of EGF receptor transcription, accompanied by a slow release in the block RNA elongation between exon 2 and exon 26, showing that EGF receptor RNA synthesis may be altered by changes in de novo transcription and by a block to RNA elongation.

Amino Acid Sequence↗
PubMed1988

Transcriptional regulation of a plasminogen activator gene by cyclic AMP in a homologous cell-free system. Involvement of cyclic AMP-dependent protein kinase in transcriptional control.

We have developed a homologous cell-free transcription system using extracts from the porcine kidney cell line LLC-PK1 to study the molecular mechanisms by which cAMP regulates urokinase-type plasminogen activator (uPA) gene transcription. We demonstrated accurate initiation of transcription using a cloned fragment of the uPA gene as template. The in vitro transcription rate was stimulated by up to 10-fold by the addition of cAMP (greater than 10 microM). This effect of cAMP on the transcription was greater for closed circular than for linear templates. Furthermore, addition of the purified catalytic subunit of cAMP-dependent protein kinase stimulated the in vitro transcription in the absence of cAMP to levels 2-fold higher than those observed with cAMP. Addition of cAMP had no stimulatory effect on the transcription of the rat heme oxygenase gene promoter tested under identical conditions. HeLa whole cell extract by itself showed no stimulation of transcription of the uPA gene by cAMP. Results of reconstitution experiments using HeLa whole cell extracts and nuclear lysates from LLC-PK1 cells suggest the presence of putative cAMP regulatory factor(s) as well as general transcription factor(s) in the nucleus of LLC-PK1 cells. These results provide experimental evidence directly implicating cAMP-dependent protein kinase in the regulation of gene transcription.

Animals↗
PubMed1992

Yeast transcription factor IID participates in cell-free transcription of a mammalian ribosomal protein TATA-less promoter.

We analysed transcription of the gene for the ribosomal protein (rp) L32 of the mouse, which is transcribed in mouse L1210 nuclear extracts in vitro. The rpL32 gene lacks a canonical TATA box. Hence it has been suggested that this gene has an alternative transcription pathway not requiring transcription factor IID (TFIID). Selective inactivation of TFIID in nuclear extract completely abolished the transcription of rpL32 in vitro. Selective inactivation was restored by the addition of cloned and purified yeast TFIID (yTFIID), indicating that this TATA-less rpL32 promoter utilizes TFIID for its transcription initiation. Furthermore, addition of an oligonucleotide-containing TATA sequence interfered with the rpL32 transcription and this was overcome by the addition of yTFIID. To further examine the stage of involvement of TFIID in rpL32 transcription, TATA oligonucleotide was added to nuclear extract before and after the formation of the transcription complex. The results reveal that TFIID associates with the pre-initiation complex and that this complex is largely resistant to added TATA oligonucleotide. Our results show, for the first time, that the TATA-less rpL32 gene utilizes TFIID for transcription initiation.

Animals↗
PubMed1988

Factors involved in specific transcription by mammalian RNA polymerase II. RNA polymerase II-associating protein 30 is an essential component of transcription factor IIF.

Transcription from class II promoters requires five general factors, IIA, IIB, IID, IIE, and IIF, in addition to RNA polymerase II for basal levels of transcription (Reinberg, D., Flores, O., and Buckbinder, L. (1987) in Molecular Biology of RNA: New Perspectives (Inouye, M., and Dudock, B., eds) pp. 423-439, Academic Press, Orlando, FL). A protein fraction containing transcription factors (TF) IIE and IIF was able to reconstitute transcription from the adenovirus major late promoter when added to extracts depleted of the RNA polymerase II-associating proteins RAP 30 and RAP 74 (Sopta, M., Carthew, R.W., and Greenblatt, J. (1985) J. Biol. Chem. 260, 10353-10360). Studies with monoaffinity-purified antibodies directed against RAP 30 demonstrated, by Western blot analysis, that RAP 30 copurifies on five columns with transcription factor IIF. That RAP 30 is a functional component of TFIIF was also demonstrated; preincubation of anti-RAP 30 antibodies with purified TFIIF inhibited transcription. Inhibition of transcription was overcome by the addition of purified TFIIF. RAP 30 is an integral part of a preinitiation complex; the incubation of all the general transcription factors with a promoter-containing DNA, prior to the addition of the anti-RAP 30 antibodies, resulted in the formation of a DNA-protein complex that was not inhibited by the antibodies. Incubation of the transcription factors in the absence of a promoter-containing DNA resulted in a complex that was partially resistant to the antibodies.

Adenoviruses, Human↗
PubMed1988

RNA processing and multiple transcription initiation sites result in transcript size heterogeneity in maize mitochondria.

Variation in the length of the 5' non-coding region of mitochondrial gene transcripts could result from multiple transcription initiation sites or post-transcriptional processing events. To distinguish between these possibilities, we have utilized the in vitro capping reaction catalyzed by guanylyl transferase to specifically label the 5' end of primary, unprocessed transcripts. Hybridization of in vitro capped mtRNA to immobilized DNA from the 5' flanking regions of 26 S, 18 S and 5 S rRNA genes and two protein-coding genes, ATP synthase subunit 9 (atp9) and apocytochrome b (cob), identified regions where transcription initiates. Single-strand specific RNase treatment of in vitro capped RNA hybridized to immobilized DNA containing the 5' flanking sequences from cob and atp9 suggests that these genes have multiple transcription initiation sites. Direct mapping of transcription initiation sites for the rRNA genes indicated that single major transcription initiation sites exist at approximately 180 and 230 nucleotides upstream from the mature 26 S and 18 + 5 S rRNA genes, respectively. Labeling of processed transcripts bearing a 5' hydroxyl moiety with T4 polynucleotide kinase and subsequent hybridization to the rRNA genes indicated that the mature forms of the rRNA are processed.

Base Sequence↗
PubMed1992

Sugarbeet minicircular mitochondrial DNAs: high-resolution transcript mapping, transcript abundance and copy number determination.

Three minicircular mitochondrial DNAs have been studied to address several aspects of transcription in sugarbeet mitochondria. High-resolution transcript mapping experiments have shown that sequences at the 5' termini of minicircle transcripts are highly homologous and resemble sequences at the 5' termini of sugarbeet mainband mitochondrial genes (atpA, atp6). In addition, they show homology to transcript termini of mitochondrial genes from other dicotyledonous plants, suggesting they may function as promoter sequences. Conserved sequences, which most probably act as RNA processing signals, were also identified at the 3' termini of minicircle transcripts. An oligonucleotide probe to a 14 base conserved sequence was used to determine the relative copy numbers of the three minicircle components in male-fertile mitochondria. Copy numbers were roughly equivalent, suggesting minicircles are replicated and/or transmitted with nearly equal efficiency, at least in sugarbeet taproots. Mc.a and Mc.c transcript levels are equivalent, consistent with their template copy number, however; Mc.d transcript levels were significantly lower than expected, implicating additional factors such as promoter strength and/or transcript stability in determining transcript levels in sugarbeet mitochondria, as recently demonstrated in maize.

Base Sequence↗
PubMed1990

The yeast heat shock transcription factor contains a transcriptional activation domain whose activity is repressed under nonshock conditions.

Transcription of heat shock genes is induced by exposure of cells to elevated temperatures or other stress conditions. In yeast, it is thought that induction of transcription is mediated by conversion of a DNA-bound transcriptionally inactive form of the heat shock transcription factor (HSTF) to a DNA-bound transcriptionally active form. We have identified domains in HSTF involved in transcriptional activation and in repression of transcriptional activation at non-shock temperatures. We present evidence that a temperature-regulated transcriptional activation domain exists in HSTF and that this domain is essential for survival of yeast cells at heat shock temperatures. We propose a model for temperature-regulated transcriptional activation by a derepression mechanism.

Amino Acid Sequence↗
PubMed1991

Heat shock transcription factor activates transcription of the yeast metallothionein gene.

In the yeast Saccharomyces cerevisiae, transcription of the metallothionein gene CUP1 is induced by copper and silver. Strains with a complete deletion of the ACE1 gene, the copper-dependent activator of CUP1 transcription, are hypersensitive to copper. These strains have a low but significant basal level of CUP1 transcription. To identify genes which mediate basal transcription of CUP1 or which activate CUP1 in response to other stimuli, we isolated an extragenic suppressor of an ace1 deletion. We demonstrate that a single amino acid substitution in the heat shock transcription factor (HSF) DNA-binding domain dramatically enhances CUP1 transcription while reducing transcription of the SSA3 gene, a member of the yeast hsp70 gene family. These results indicate that yeast metallothionein transcription is under HSF control and that metallothionein biosynthesis is important in response to heat shock stress. Furthermore, our results suggest that HSF may modulate the magnitude of individual heat shock gene transcription by subtle differences in its interaction with heat shock elements and that a single-amino-acid change can dramatically alter the activity of the factor for different target genes.

Amino Acid Sequence↗
PubMed1992

The basic region of myogenin cooperates with two transcription activation domains to induce muscle-specific transcription.

Myogenin is a skeletal muscle-specific transcription factor that can activate myogenesis when introduced into a variety of nonmuscle cell types. Activation of the myogenic program by myogenin is dependent on its binding to a DNA sequence known as an E box, which is associated with numerous muscle-specific genes. Myogenin shares homology with MyoD and other myogenic regulatory factors within a basic region and a helix-loop-helix (HLH) motif that mediate DNA binding and dimerization, respectively. Here we show that the basic region-HLH motif of myogenin alone lacks transcriptional activity and is dependent on domains in the amino and carboxyl termini to activate transcription. Analysis of these N- and C-terminal domains through creation of chimeras with the DNA-binding domain of the Saccharomyces cerevisiae transcription factor GAL4 revealed that they act as strong transcriptional activators. These transcription activation domains are dependent for activity on a specific amino acid sequence within the basic region, referred to as the myogenic recognition motif (MRM), when an E box is the target for DNA binding. However, the activation domains function independent of the MRM when DNA binding is mediated through a heterologous DNA-binding domain. The activation domain of the acidic coactivator VP16 can substitute for the myogenin activation domains and restore strong myogenic activity to the basic region-HLH motif. Within a myogenin-VP16 chimera, however, the VP16 activation domain also relies on the MRM for activation of the myogenic program. These findings reveal that DNA binding and transcriptional activation are separable functions, encoded by different domains of myogenin, but that the activity of the transcriptional activation domains is influenced by the DNA-binding domain. Activation of muscle-specific transcription requires collaboration between the DNA-binding and activation domains of myogenin and is dependent on events in addition to DNA binding.

Amino Acid Sequence↗
PubMed1988

Transcription fraction TFIIIC can regulate differential Xenopus 5S RNA gene transcription in vitro.

An extract of whole oocytes (oocyte S150) differentially transcribes Xenopus oocyte and somatic 5S RNA genes. In the oocyte S150, transcription complexes with different stabilities are assembled onto oocyte and somatic 5S DNA. The stability of the transcription complex is dependent on activities present in a fraction containing transcription factor TFIIIC. This fraction stabilizes the binding of the positive transcription factor TFIIIA to a somatic 5S RNA gene much more efficiently than it does to an oocyte gene. The oocyte S150 transcription extract is deficient in TFIIIC such that supplementation with a fraction enriched in this transcription factor selectively stimulates oocyte 5S DNA transcription. Previously it has been shown that an egg extract deficient in TFIIIA selectively transcribes somatic 5S RNA genes. Thus under conditions where there is differential stability of transcription complexes, limitation of either TFIIIA or TFIIIC may exaggerate the differential expression of two genes.

Animals↗
PubMed1988

Light-dependent changes in psbD and psbC transcripts of barley chloroplasts: accumulation of two transcripts maintains psbD and psbC translation capability in mature chloroplasts.

The psbD and psbC genes encode two polypeptides of Photosystem II. These genes are adjacent in the barley chloroplast genome and are part of a 5.7 kbp transcription unit. In dark-grown barley, four large transcripts hybridize to psbD and psbC; two additional transcripts hybridize to psbC. Illumination of 4.5-day-old dark-grown seedlings causes a decrease in the six psbD--psbC transcripts found in etioplasts and the accumulation of two different transcripts of 4.0 and 3.2 kb which hybridize to psbD and psbC. The light-induced transcripts have a common 5' end approximately 600 nt upstream of psbD and 3' ends 1175 and 175 nt downstream of psbC. The shift in psbD--psbC transcript population occurs during a phase of chloroplast maturation when transcript levels and translation of chloroplast genes such as psaA--psaB and psbB decline approximately 3- to 5-fold. In contrast, translation of the psbD and psbC gene products declines to a lesser extent, suggesting that the light-induced accumulation of the 4.0 and 3.2 kb psbD--psbC transcripts is required to maintain psbD and psbC gene product translation in mature chloroplasts.

Chlorophyll↗
PubMed1991

Faithful in vivo transcription termination of Xenopus laevis rDNA. Correlation of electron microscopic spread preparations with S1 transcript analysis.

DNA sequencing and subsequent functional in vitro analysis of the Xenopus laevis rDNA transcription termination has led to the identification of three transcription termination sequence elements: T1, located at the 3' end of the 28S rDNA; T2, a putative processing site 235 bp downstream of T1; T3, the principal terminator positioned 215 bp upstream of the gene promoter. As demonstrated for nuclear run-off assays, T3 was found to be the main terminator for Xenopus rDNA transcription. These in vitro data are in obvious contradiction to results obtained by electron microscopic (EM) spread preparations from rapidly isolated amplified oocyte nucleoli, i.e., an rDNA chromatin probe thought to represent the in vivo situation, indicative of transcription termination at sites T1-2. However, most interestingly, T3 had--again by the EM method--been identified as the exclusive terminator for NTS spacer transcription units. In order to answer the question of whether read-through transcription of the complete rDNA spacer sequence is obligatory for 40S pre-rRNA in vivo transcription, we analyzed several hundreds of spread rRNA genes from Xenopus oocyte nucleoli in great detail, applying two different spreading procedures, e.g., dispersal of amplified oocyte nucleoli shortly in detergent-free or detergent containing low-salt media prior to the EM spreading technique. Quantitation of EM spreads resulted in the finding that read-through rDNA spacer transcription beyond T1-2 termination sites (i.e., indicative of T3 transcription termination) can be visualized for the in vivo situation at a frequency of less than 3% of rRNA genes analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Self-transcription: RNA polymerase transcription of its own genes, and its role in cellular differentiation and cell cycling.

The concept of RNA polymerase self-transcription, where eukaryotic RNA polymerase II and prokaryotic holoenzyme are responsible for transcription of their own genes, would give these enzymes a unique role in the cellular transcriptional and translational machinery. This self-transcriptional ability would equip a cell with an exquisite mechanism of autogenous regulation for the appearance of these transcriptional units. Such a mechanism could allow layering of other transcriptional control pathways upon the RNA polymerase self-transcriptional pathway, thus forming a complex array of mechanisms to regulate transcription of genes concerned with cellular differentiation and cell cycling. It is proposed that RNA polymerase self-transcription is the central control point of gene expression in cellular differentiation and for cell cycling, thus fulfilling the role of an intrinsic biological clock.

Animals↗
PubMed1989

Transcription map of the B genome component of tomato golden mosaic virus and comparison with A component transcripts.

In a previous study, the bipartite genome of tomato golden mosaic virus (TGMV) was shown to be transcribed into at least six polyadenylated RNAs (G. Sunter, W.E. Gardiner, and D. M. Bisaro, 1989, Virology 170, 243-250). Two of these, a 1.3-kb complementary sense and a 0.9-kb viral sense transcript, were mapped to the B genome component of this geminivirus. The results of more detailed primer extension and S1 nuclease protection experiments presented here define the limits of the single transcription unit corresponding to the 0.9-kb RNA which spans the BR1 open reading frame (ORF). The data also demonstrate that complementary sense TGMV RNAs are more complex than indicated by our earlier studies. Analysis of the 1.3-kb BL1-specific RNA indicates that it is actually a family of distinct transcripts with different start sites. Three transcripts have 5' ends that map near the common region of DNA B and all of these start sites lie upstream of the BL1 ORF. Similar analysis of the 1.6-kb complementary sense AL1 RNA indicates that a complex set of transcripts also map to the analogous region of genome component A. Four transcripts have 5' ends that map near the common region but only one of these start sites is upstream of the initiation codon for the AL1 open reading frame (ORF). None of the transcripts appear to be processed. The possible significance of multiple transcripts in these regions of the TGMV genome is discussed, and the common region-proximal transcription units of the A and B genome components are compared.

Autoradiography↗
PubMed1990

Effects of nutrients and hormones on transcriptional and post-transcriptional regulation of fatty acid synthase in rat liver.

The effects of nutrients and hormones on transcriptional and post-transcriptional regulation of fatty acid synthase in rat liver were investigated following cDNA cloning. When fasted rats were fed a carbohydrate/protein diet, the transcriptional rate was greatly increased even in 1 h. The transcriptional rate, mRNA concentration and enzyme induction reached maximum levels in 4 h, 8-16 h and 48 h, respectively. Although dietary carbohydrate increased each level more than protein did, both carbohydrate and protein were required to reach a high level. Corn oil feeding markedly decreased the transcriptional rate. In diabetic rats, the transcriptional rate, mRNA concentration and enzyme induction were very low in comparison with the normal. By treating the diabetic rats with insulin, however, the transcriptional rate was increased 5-fold in 1 h and 15-fold in 6 h, preceding a great increase in the mRNA and enzyme levels. On the other hand, fructose feeding or triiodothyronine treatment of diabetic rats abundantly increased the mRNA concentration and somewhat increased the transcriptional rate. Thus, it is suggested that insulin mainly stimulates the transcription of the fatty acid synthase gene, whereas triiodothyronine and fructose mainly increase the mRNA stability.

Animals↗
PubMed1981

Transcriptional and post-transcriptional control of ribosomal protein and ribonucleic acid polymerase genes.

A partial restriction of ribonucleic acid (RNA) polymerase activity has been used to dissociate the coordinate synthesis of ribosomal proteins and subunits of RNA polymerase and to identify transcriptional and post-transcriptional control signals which regulate the expression of these component genes. Within the beta operon [which has the genetic organization: promoter (p beta), rplJ (L10), r;lL (L7/L12), attenuator, rpoB (beta), rpoC (beta'), terminator], the restriction caused a disproportionate increase between proximal and distal gene transcriptions; the transcriptional intensities of the proximal ribosomal protein genes and the distal RNA polymerase genes were elevated about two- and fourfold, respectively. Transcription within the operon containing four ribosomal protein genes and the RNA polymerase alpha gene was also enhanced, whereas transcription within operons containing only ribosomal protein genes was virtually unaffected by the restriction. It was thus concluded that the mechanisms controlling transcription initiation or attenuation or both in operons containing RNA polymerase subunit genes are coupled to the global rate of RNA synthesis. By introducing the composite ColE1 plasmid pJC701 carrying the proximal portion of the L10 operon, including the beta subunit gene, it was possible to achieve a 10- and a 30-fold range in the transcriptional intensities of the genes specifying L10 and L7/L12 and beta, respectively. Under these conditions, the relative synthesis rates of L7/L12 and beta protein varied by less than 2-fold and by about 15-fold, respectively. These observations corroborate the existence of a post-transcriptional mechanism which severely restricts translation of excess L7/L12 and L10 ribosomal protein messenger RNA; this mechanism is probably important in maintaining the balanced synthesis of ribosome components under conditions in which their messenger RNA levels are dissociated. Furthermore, the observed reduction in the translation efficiency of beta subunit messenger RNA may be related to an inhibitory effect caused by accumulation of RNA polymerase assembly intermediates.

Bacterial Proteins↗
PubMed1991

Adenovirus E1A protein activates transcription of the E1A gene subsequent to transcription complex formation.

The mechanism of transcriptional activation of the adenovirus E1A and E3 genes by E1A protein during infection was examined by using transcription-competition assays. Infection of HeLa cells with one virus led to inhibition of mRNA accumulation from a superinfecting virus. Synthesis of the E1A 289R protein by the first virus to infect reduced inhibition of transcription of the superinfecting virus, indicating that the E1A 289R protein was limiting for E1A-activated transcription. Infection with an E1A- virus, followed 6 h later by superinfection with a wild-type virus, led to preferential transcriptional activation of the E1A gene of the first virus, suggesting that a host transcription component(s) stably associated with the E1A promoter in the absence of E1A protein and that this complex was the substrate for transcriptional activation by E1A protein. The limiting host transcription component(s) bound to the E1A promoter to form a complex with a half-life greater than 24 h in the absence of E1A 289R protein, as demonstrated in a challenge assay with a large excess of superinfecting virus. In the presence of the E1A 289R protein, the E1A gene of the superinfecting virus was gradually activated with a reduction in E1A mRNA accumulation from the first virus. The kinetics of the activation suggest that this was due to an indirect effect rather than to destabilization of stable transcription complexes by the 289R protein.

Adenovirus Early Proteins↗
PubMed1986

Adenovirus stimulation of transcription by RNA polymerase III: evidence for an E1A-dependent increase in transcription factor IIIC concentration.

Human cells expressing adenovirus E1A proteins transcribe transfected tRNA and adenovirus VAI genes at greater than 10-fold higher levels than uninfected HeLa cells. Here we show that the increased transcription observed in vivo is reflected in the in vitro transcriptional activity of cell extracts. Depletion of E1A protein from these extracts by immunoprecipitation with a monoclonal antibody did not diminish the activity, suggesting that E1A proteins do not stimulate transcription directly. Fractionation of the extracts by chromatography on phosphocellulose suggests that the higher activity of extracts of adenovirus-infected cells was due to increased activity of the transcription factor (TF) which is the limiting component required for specific initiation of tRNA and VAI transcription in extracts of uninfected HeLa cells, i.e. TFIIIC. Template commitment titrations further suggest that the increased TFIIIC activity was due to an increase in the concentration of active TFIIIC. On the basis of these results and recent genetic analyses of early adenovirus promoters, we suggest that E1A proteins stimulate transcription of adenovirus genes indirectly by increasing the effective in vivo concentration of the limiting cellular transcription factors required for their transcription.

Adenovirus Early Proteins↗