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At least 109 records · Page 6Linked to original sources
PubMed1991

Transcriptional and post-transcriptional regulation of 72-kDa gelatinase/type IV collagenase by transforming growth factor-beta 1 in human fibroblasts. Comparisons with collagenase and tissue inhibitor of matrix metalloproteinase gene expression.

The temporal aspects and mechanisms of the regulation of the matrix metalloproteinase (MMP) 72-kDa gelatinase/type IV collagenase (MMP-2) by transforming growth factor-beta 1 (TGF-beta 1) were investigated in early passage human gingival fibroblasts and compared with the regulation of the genes for collagenase (MMP-1) and TIMP, the tissue inhibitor of MMPs. Northern hybridization analyses revealed that 1.0 ng/ml TGF-beta 1 increased the abundance of MMP-2 mRNA/cell approximately 1.5-fold at 24 h, an increase similar to that observed in the level of [35S]methionine pulse-labeled MMP-2 at 24 h (1.9-fold). At 48 and 72 h, the increase in MMP-2 mRNA abundance remained elevated by 1.5-2.2-fold on a per cell basis whereas TIMP mRNA levels were elevated by up to 3.3-fold. In contrast, the relative levels of collagenase mRNA were reduced by 66-75%. The changes in the MMP-2, collagenase, and TIMP mRNA concentrations in response to TGF-beta 1 were blocked by cycloheximide indicating that protein synthesis was required to mediate the effects of TGF-beta 1 on these mRNA levels. TGF-beta 1 was also found to increase the half-life of the MMP-2 mRNA from approximately 46 to approximately 150 h but did not alter the stability of TIMP mRNA (t1/2 approximately 60 h). Nuclear run-off transcription assays revealed that MMP-2 gene transcription was increased approximately 5-fold 7 h following TGF-beta 1-treatment but returned to control levels by 24 h. In comparison, increased TIMP gene transcription was only detectable after 24 h whereas collagenase gene transcription, although low in control cells, was undetectable at 24 h. Gene transcription, mRNA levels, and message stability of the genes for the extracellular matrix proteins type I collagen and fibronectin were also increased by TGF-beta 1. Thus, the similarity in the control of MMP-2, alpha 1 (I) procollagen, and fibronectin expression at the transcriptional and post-transcriptional levels indicates that these genes may share regulatory elements. In comparison, TGF-beta 1 reduced the level of collagenase mRNA and increased the level of TIMP mRNA as a result of altered transcriptional activities, through pathways that required protein synthesis, and without changes in mRNA stability.

Blotting, Northern↗
PubMed1983

Mutations of the beta subunit of RNA polymerase alter both transcription pausing and transcription termination in the trp operon leader region in vitro.

RNA polymerase was purified from rifampicin-resistant mutants of Escherichia coli which exhibit altered transcription termination at the trp operon attenuator in vivo. These mutant polymerases were used to investigate transcription pausing at the trp leader pause site and transcription termination at the trp attenuator. The mutant polymerases examined in vitro mimic their in vivo termination responses; i.e. RNA polymerase isolated from a mutant which displays high transcriptional read-through of the trp operon in vivo allows greater transcriptional read-through in vitro, while RNA polymerase prepared from a mutant which has reduced read-through in vivo exhibits greater termination of transcription in vitro. The observed differences are not due to the presence of--or response to--alternate secondary structures in the trp leader transcript since deletion of the DNA segment corresponding to some of these alternate structures does not affect termination efficiency. The mutant polymerases also have comparable effects on the kinetics of transcription pausing at the trp leader pause site; the termination-deficient polymerase exhibits diminished pausing while the termination-proficient polymerase displays enhanced pausing. We suggest that this correlation reflects polymerase recognition of similar features of RNA secondary structures in the pause and termination events. In addition, since single mutational changes in RNA polymerase affect two activities, pausing and termination, it is likely that a single site or region of the polymerase is involved in both events.

Base Sequence↗
PubMed1990

Engrailed, a homeodomain protein, can repress in vitro transcription by competition with the TATA box-binding protein transcription factor IID.

Engrailed (En) is a homeodomain protein that binds to a consensus sequence (NP) and plays an important role during Drosophila development. Purified En, which is produced in Escherichia coli, binds not only to this consensus sequence but also to the TATA box of the Drosophila Hsp70 promoter and of other eukaryotic promoters. Interestingly, En represses transcription of these promoters in an in vitro-reconstituted mammalian transcription system and footprint analyses show that En competes with the TATA box-binding protein transcription factor IID for binding to the TATA box. In contrast, a stable template-committed complex formed by preincubation of transcription factor IID with the promoter is not disrupted by addition of En, and in this case transcription is not repressed. These in vitro studies suggest a transcriptional repression mechanism, involving competition between En and transcription factor IID for TATA box binding, that may be involved in En-mediated repression in vivo.

Animals↗
PubMed1988

An RNA polymerase II transcription factor inactivated in poliovirus-infected cells copurifies with transcription factor TFIID.

Inhibition of host cell RNA polymerase II-mediated transcription by poliovirus infection was studied in vitro. Whole-cell extracts prepared from poliovirus-infected HeLa cells at 3 h postinfection were shown to be deficient in a factor required for specific transcription from the adenovirus major late promoter. Three lines of evidence suggest that transcription factor TFIID is deficient in poliovirus-infected cells. First, the activity required to specifically restore transcription in poliovirus-infected cell extracts was shown to copurify with TFIID through three chromatographic steps. Second, transcription reactions reconstituted with phosphocellulose-derived chromatographic fractions revealed a fourfold decrease in the specific activity of the TFIID-containing fraction prepared from poliovirus-infected cells compared with that of the same fraction prepared from mock-infected cells. Finally, TFIID and the activity required to specifically restore transcription in virus-infected cell extracts were shown to have the same kinetics of heat inactivation. Together, these results suggest that inactivation of TFIID is an early event in the inhibition of host cell RNA polymerase II transcription by poliovirus.

Cell Transformation, Viral↗
PubMed1990

Transcriptional and post-transcriptional regulation of c-jun expression during monocytic differentiation of human myeloid leukemic cells.

AP-1, the polypeptide product of c-jun, recognizes and binds to specific DNA sequences and stimulates transcription of genes responsive to certain growth factors and phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA). We studied the effects of TPA on the regulation of c-jun gene expression in HL-60 cells during monocytic differentiation. Low levels of c-jun transcripts were detectable in untreated HL-60 leukemic cells, increased significantly by 6 h, and reached near maximal levels by 24 h of exposure to 32 nM TPA. Similar kinetics of c-jun induction by TPA were observed in human U-937 and THP-1 monocytic leukemia cells. Similar findings were obtained with bryostatin 1 (10 nM), another activator of protein kinase C and inducer of monocytic differentiation. Furthermore, 1,25-dihydroxyvitamin D3 (0.5 microM), a structurally distinct agent which also induces HL-60 monocytic differentiation, increased c-jun expression. TPA treatment of HL-60 cells in the presence of cycloheximide was associated with superinduction of c-jun transcripts. Run-on analysis demonstrated detectable levels of c-jun gene transcription in untreated HL-60 cells, and that exposure to TPA increases this rate 3.3-fold. Treatment of HL-60 cells with both TPA and cycloheximide had no effect on the rates of c-jun transcription. The half-life of c-jun RNA as determined by treating HL-60 cells with TPA and actinomycin D was 30 min. In contrast, the half-life of c-jun RNA in TPA-treated HL-60 cells exposed to cycloheximide and actinomycin D was greater than 2 h. These findings suggested that the increase in c-jun RNA observed during TPA-induced monocytic differentiation is mediated by both transcriptional and post-transcriptional mechanisms.

Calcitriol↗
PubMed1990

Sarkosyl defines three intermediate steps in transcription initiation by RNA polymerase III: application to stimulation of transcription by E1A.

We used Sarkosyl to analyze steps along the pathway of transcription initiation by RNA polymerase III. Sarkosyl (0.015%) inhibited transcription when present prior to incubation of RNA polymerase III, TFIIIB, and TFIIIC with the VAI gene, whereas it had no detectable effect on initiation or reinitiation of transcription when added subsequently. The formation of the corresponding 0.015% Sarkosyl-resistant complex required the presence of TFIIIC, TFIIIB, and RNA polymerase III but not nucleoside triphosphates. The addition of 0.05% Sarkosyl after this early step selectively inhibited a later step in the preinitiation pathway, allowing a single round of transcription after nucleoside triphosphate addition but blocking subsequent rounds of initiation. This step occurred prior to initiation because nucleoside triphosphates were not required for the formation of the corresponding 0.05% Sarkosyl-resistant complex. These observations provided a means to distinguish effects of regulatory factors on different steps in promoter activation and function. Using 0.05% Sarkosyl to limit reinitiation, we determined that the E1A-mediated stimulation of transcription by RNA polymerase III resulted from an increase in the number of active transcription complexes.

Adenovirus Early Proteins↗
PubMed1991

Analysis of the molecular mechanisms for the species-specific transcription of Drosophila and human tRNA gene transcription components.

The transcription of eucaryotic tRNA genes requires two factors IIIB and IIIC, in addition to RNA polymerase III, to reconstitute this process in vitro. We have examined the functional exchangeability of these components from Drosophila and human systems. The reconstitution of heterologous IIIB and IIIC components demonstrated that neither factor will functionally substitute for the homologous components to activate tRNA gene transcription. The addition of the heterologous Drosophila factors to HeLa transcription assays causes an inhibition of RNA synthesis that is dependent upon the order of addition of these proteins to the DNA template. Thus, it appears that tRNA gene transcription in these systems is species-specific. We have further analyzed the reason for the apparent incompatibilities of these components by the use of stable complex formation assays. We find that human HeLa IIIB and Drosophila IIIC are unable to form stably associated complexes with a tRNA gene template, whereas the Drosophila IIIB and HeLa IIIC do form stable but nonproductive complexes. These results demonstrate that specific IIIC-IIIB interactions are critical in the formation of productive transcription complexes and are responsible for the observed species specificity of Drosophila and human tRNA gene transcription.

Animals↗
PubMed1984

Expression of the clustered mitochondrial tRNA genes in Saccharomyces cerevisiae: transcription and processing of transcripts.

The transcripts of a cluster of eight tRNA genes localized in the Cap-oxiI region of the mitochondrial genome of Saccharomyces cerevisiae were investigated by hybridization of gene-specific probes on Northern blots of mitochondrial RNA and by S1 mapping of the 5' termini of the transcripts. Two rho- mutants that lack mature tRNA species and accumulate precursors have been used to detect transcripts that are not detectable in wild-type (w.t.) mitochondria. The results have shown the existence of polygenic transcripts carrying at least 5-7 tRNA sequences, both in w.t. and in rho- strains. The existence of several alternative processing pathways, which involve cleavage at the 3' and 5' ends of the tRNA sequences and in the long intergenic regions (possibly at GC clusters), is suggested. Cleavage at the 5' ends of tRNA sequences is defective in the mutant strains. The transcripts of the genes for tRNAThrACN and tRNACys (the tRNA genes immediately downstream from the 21S rRNA gene) have been analyzed; the possibility that these species represent primary transcripts is considered, and potential sites for initiation of transcription of the clustered tRNA genes are discussed.

Base Sequence↗
PubMed1987

Hepatoma variants (C2) are defective for transcriptional and post-transcriptional actions from both endogenous and viral genomes.

A series of rat hepatoma cell lines was infected with a recombinant adenovirus bearing the rat albumin promoter. Transcription from this promoter was scored directly and was highest in FAO, the differentiated parent, undetectable in C2, a cell variant that has lost almost all hepatocytic characteristics, and high again in C2-Rev7, a 'revertant' cell line derived from C2 that has regained the ability to produce many proteins characteristic of hepatocytes. The endogenous albumin gene is not transcribed in C2 cells, and at a very low rate in C2-Rev7 cells, which accumulate endogenous albumin mRNA at close to normal amounts. Thus the C2-Rev7 'recovery' of albumin mRNA concentration for the endogenous gene is based mainly on post-transcriptional events while the ability of C2-Rev7 to transcribe the albumin promoter in the viral genome is based on a transcriptional factor(s). We also showed that the C2 phenotype included post-transcriptional effects for other genes: transcription of phenylalanine hydroxylase and phosphoenolpyruvate carboxykinase mRNA sequences continue in C2 at rates equivalent to FAO but these C2 cells have no mRNA for these proteins while FAO does. In addition, C2 cells transcribed certain early adenovirus transcription units (E2 and 4) as well as FAO cells but accumulated E2 mRNAs poorly if at all. The changes that led to the C2-Rev7 cell line produced a return to normal of the ability to accumulate these viral mRNAs. Thus a major event in the C2 to C2-Rev7 transition involves post-transcriptional processes as well as the ability to transcribe the albumin promoter positioned in the virus genome.

Adenoviridae↗
PubMed1986

Yeast class III gene transcription factors and homologous RNA polymerase III form ternary transcription complexes stable to disruption by N-lauroyl-sarcosine (sarcosyl).

Yeast Class III gene transcription factors and RNA polymerase III were used to form ternary transcription complexes on a tRNASer gene in vitro under UTP-limiting transcription conditions. These ternary transcription complexes were composed of template DNA, proteins, and RNA. We have shown that the RNAs contained in these complexes represented specifically initiated nascent pre-tRNASer transcripts. These nascent RNAs could be very efficiently elongated to full-length pre-tRNASer molecules, even in the presence of the ionic detergent sarcosyl. Partial purification (greater than 100-fold) of these sarcosyl-resistant ternary transcription complexes could be achieved in a single step via sucrose gradient sedimentation. Comparable sarcosyl-resistant ternary transcription complexes could not be formed using purified yeast RNA polymerase III as the only protein component of the complex.

Base Sequence↗
PubMed1982

Transcription of E. coli and Euglena chloroplast tRNA gene clusters and processing of polycistronic transcripts in a HeLa cell-free system.

The transcription of cloned tRNA genes that are clustered in the Escherichia coli and Euglena gracilis chloroplast genome was studied in a HeLa cell-free extract. RNA polymerase III transcribes the tRNA gene clusters into polycistronic primary transcripts, consisting of pre-tRNAVal-tRNAAsn-tRNAArg and pre-tRNAAsp-tRNATrp transcribed from the Euglena chloroplast and E. coli tRNA locus, respectively. A UV5-lac promoter in the 5'-flanking sequence of the E. coli tRNA gene cluster can be removed without effect on transcription initiation frequency. During transcription of the tRNA gene clusters, RNA polymerase III initiates predominantly, if not solely, with the first tRNA gene. Upon removal of the first tRNA gene, RNA polymerase III initiates transcription with the following tRNA gene. The primary transcripts are subsequently processed to mature tRNAs. RNA fingerprint analysis and reincubation experiments were used to establish the steps of tRNA maturation of the pre-tRNAVal-tRNAAsn-tRNAArg. The 3' trailer is processed prior to the removal of 5' leader and before cleavage of the primary transcript in the spacer sequences. The pre-tRNAs are then processed to mature tRNA molecules.

Cell-Free System↗
PubMed1989

Purified octamer binding transcription factors stimulate RNA polymerase III--mediated transcription of the 7SK RNA gene.

We have analyzed the upstream promoter of the human 7SK RNA gene to determine which protein factors are involved in the transcription of this gene by RNA polymerase III. Using a reconstituted in vitro system, we show directly that octamer binding transcription factors (OTFs) are required for efficient transcription and that they interact with a series of nonconsensus OTF binding sites between positions -70 and -240. The same purified factors that stimulate RNA polymerase II-dependent transcription of the histone H2b gene (OTF-1) and an immunoglobulin light chain gene (OTF-2) also stimulate 7SK transcription by RNA polymerase III. Moreover, OTF-dependent stimulation requires a sequence between positions -48 and -65 that is homologous to the proximal sequence element of the class II snRNA genes. Our findings indicate that some transcription factors are utilized in the transcription of both class II and class III genes.

Base Sequence↗
PubMed1986

Transcriptional and post-transcriptional inhibition of albumin gene expression by estrogen in Xenopus liver.

The purpose of this study was to investigate the suppression of albumin mRNA by estrogen in Xenopus liver. A single dose of estradiol rapidly suppressed albumin mRNA to 30% of the control level. Albumin mRNA remained at this new steady-state level for 9 days, after which it returned to the control level. Transcription 'run-on' experiments in isolated liver nuclei demonstrated a transient decrease of 60-90% in albumin transcription after 2-6 h and approached constitutive transcription by 12 h. Albumin gene transcription then remained constant for the following 12 days. Prolonged and enhanced suppression of albumin mRNA was observed in animals treated repeatedly with estrogen for 12 days. In these animals, albumin gene transcription was decreased 80-90% from the constitutive control level. These data indicate that albumin mRNA is suppressed by both transcriptional and post-transcriptional mechanisms.

Albumins↗
PubMed1982

Function of transcription termination factor rho in a model transcription system using synthetic deoxyribonucleic acid as template.

The function of a transcription termination factor, rho, has been studied by using several synthetic DNAs with simple repetitive base sequences as templates for transcription. rho actually exhibits various effects on transcription depending on the base sequence of the template: (1) rho terminates poly(A) synthesis with poly(dA) x poly(dT), poly(dT), or oligo(dT), leading to release of RNA from RNA polymerase. rho also inhibits the synthesis of other homoribopolymers such as poly(U) directed by poly(dA) x poly(dT) and poly(C) and poly(I) directed by poly(dG) x poly(dC), presumably by a similar mechanism. (2) rho inhibits the synthesis of another homoribopolymer, poly(G), directed by poly(dG) x poly(dC) at the step of initiation rather than propagation of transcription. (3) rho stimulates rather than inhibits the synthesis of poly(A-C) and poly(G-U) directed by poly[d(A-C)] x poly[d(G-T)], presumably by enhancing the dissociation of transcription complexes. (4) rho has no influence on the synthesis of poly(A-U) and poly(G-C) directed by poly[d(A-T)] and poly[d(G-C)], respectively. In the first case, but not otherwise, the effect of rho is coupled with its RNA-dependent nucleosidetriphosphate phosphohydrolase activity, as is rho-mediated transcription termination on natural templates. The implication of these results is discussed in reference to the current view that rho acts on transcription complexes that have ceased elongation and causes release of RNA in an energy-requiring reaction.

Base Sequence↗
PubMed1988

The JUN oncoprotein, a vertebrate transcription factor, activates transcription in yeast.

Transcriptional activation of RNA polymerase II in eukaryotic organisms ranging from yeasts to mammals has many common features such as enhancer elements, TATA elements, and activator proteins that bind specifically to promoter DNA. The JUN oncoprotein, which causes sarcomas in chickens, shows significant homology to the DNA-binding domain of GCN4, a yeast protein that stimulates transcription of the amino acid biosynthetic genes. The GCN4 and JUN proteins bind the same DNA sequences, consensus ATGA(C/G)TCAT, even though the DNA-binding domains are only 45% identical in amino acid sequence. The JUN protein almost certainly represents the oncogenic version of the normal AP-1 transcription factor, suggesting an evolutionary relationship between yeast and vertebrate activator proteins. Here, I demonstrate that JUN efficiently activates transcription in yeast either through its own or a heterologous DNA-binding domain. As is the case for yeast activator proteins, transcriptional stimulation by JUN requires an acidic activation region distinct from the DNA-binding domain. The functional interchangeability between yeast and vertebrate transcription factors strongly suggests a basic similarity in the molecular mechanism of eukaryotic transcriptional activation.

Amino Acid Sequence↗
PubMed1991

Transcriptional interference between the EBV transcription factors EB1 and R: both DNA-binding and activation domains of EB1 are required.

The switch from latency to a productive infection in EBV-infected B cells is linked to the expression of two viral sequence-specific DNA-binding transcription factors called EB1 and R. EB1 shares sequence homologies with the bZIP family of proteins in the basic region required for specific DNA interaction. Here, we provide evidence that EB1 and R can synergistically activate specific transcription, and that overexpressed, unbound EB1, represses the R-induced transcription ('squelching'). In order to identify the EB1 domains involved in transcriptional activation, transcriptional synergy and transcriptional repression, we performed extensive mutagenesis of the EB1 protein. Results show that five segments (region 1 to region 5), localized at the N-terminus of EB1 exhibit characteristics of activating domains, since they are required for full transcriptional activity, without obvious role in DNA-binding, or the nuclear localization. Two domains rich in basic amino-acids are required for the nuclear localization of EB1. One domain is within the basic region B, also necessary for specific and stable interaction between EB1 and its cognate DNA sequences. It is also shown that the 'activation' domain, and more surprisingly the DNA-binding domain of EB1, may interact with a factor(s), essential for R-induced activation, and probably required for synergy between EB1 and R.

Amino Acid Sequence↗
PubMed1990

Single cell assay of a transcription factor reveals a threshold in transcription activated by signals emanating from the T-cell antigen receptor.

Stimulation of T lymphocytes through their antigen receptor leads to the appearance of several transcription factors, including NF-AT and NF-kappa B, which are involved in regulating genes required for immunologic activation. To investigate the activity of a single transcription factor in individual viable cells, we have applied an assay that uses the fluorescence-activated cell sorter to quantitate beta-galactosidase (beta-gal). We have analyzed the distribution of NF-AT transcriptional activity among T cells undergoing activation by using a construct in which three tandem copies of the NF-AT-binding site directs transcription of the lacZ gene. Unexpectedly, stimulation of cloned stably transfected Jurkat T cells leads to a bimodal pattern of beta-gal expression in which some cells express no beta-gal and others express high levels. This expression pattern cannot be accounted for by cell-cycle position or heritable variation. Further results, in which beta-gal activity is correlated with NF-AT-binding activity, indicate that the concentration of NF-AT must exceed a critical threshold before transcription initiates. This threshold likely reflects the NF-AT concentration-dependent assembly of transcription complexes at the promoter. Similar constructs controlled by NF-kappa B or the entire interleukin-2 enhancer show bimodal expression patterns during induction, suggesting that thresholds set by the concentration of transcription factors may be a common property of inducible genes.

Cell Cycle↗
PubMed1985

Mitochondrial transcription and processing of transcripts during release from glucose repression in 'resting cells' of Saccharomyces cerevisiae.

Mitochondrial transcription and processing of transcripts have been investigated at different stages of release from glucose repression in resting cells of Saccharomyces cerevisiae. Transcripts were identified by hybridization with nick-translated or terminally labelled gene-specific probes. This allowed the determination of the steady-state levels of individual transcripts in the mitochondrial RNA population. Results showed different gene-specific patterns of response to respiratory induction: no increase in the level of transcripts (oxi2); a rapid increase in the steady-state levels of all transcripts (cob); a very strong increase in the processing of the high-molecular-mass precursors (oxi3 and oli2); an increase in the level of stable circular transcripts (oxi3). As a whole the results indicate specific and differentiated effects of release from glucose repression on the expression of the different mitochondrial genes and demonstrate the importance of processing events in mitochondrial regulation.

Autoradiography↗