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At least 19 recordsLinked to original sources
PubMed1989

Bovine mitochondrial protein synthesis elongation factors. Identification and initial characterization of an elongation factor Tu-elongation factor Ts complex.

Animal mitochondrial protein synthesis factors elongation factor (EF) Tu and EF-Ts have been purified as an EF-Tu.Ts complex from crude extracts of bovine liver mitochondria. The mitochondrial complex has been purified 10,000-fold to near homogeneity by a combination of chromatographic procedures including high performance liquid chromatography. The mitochondrial EF-Tu.Ts complex is very stable and cannot be dissociated even in the presence of high concentrations of guanine nucleotides. No guanine nucleotide binding to this complex can be observed in the standard nitrocellulose filter binding assay. Mitochondrial EF-Ts activity can be detected by its ability to facilitate guanine nucleotide exchange with Escherichia coli EF-Tu. The EF-Tumt exhibits similar levels of activity on isolated mammalian mitochondrial and E. coli ribosomes, but displays minimal activity on Euglena gracilis chloroplast 70 S ribosomes and has no detectable activity on wheat germ cytoplasmic ribosomes. In contrast to the bacterial EF-Tu and the EF-Tu from the chloroplast of E. gracilis, the ability of the mitochondrial factor to catalyze polymerization is not inhibited by the antibiotic kirromycin.

Animals↗
PubMed2026

Targeting super elongation complex-driven RNA polymerase II elongation reduces plaque vulnerability.

Atherosclerotic plaque rupture is a major cause of myocardial infarction and stroke, yet the mechanisms governing plaque stability remain incompletely understood. Endothelial activation can trigger endothelial-to-mesenchymal transition, a program linked to endothelial dysfunction and lesion vulnerability. Here we investigated whether transcriptional pause release and RNA polymerase II elongation constitute an early regulatory layer that promotes endothelial-to-mesenchymal transition and atherosclerosis. Analysis of human plaque single-cell transcriptomics indicated increased expression of super elongation complex components in endothelial cells with a transition signature. In primary human endothelial cell models, pharmacological inhibition of the super elongation complex attenuated the induction of mesenchymal markers. AFF4, pCDK9, and pSMAD2/3 showed physical interaction during endothelial transition. Genome-wide profiling of RNA polymerase II occupancy revealed reduced promoter-proximal pausing during early transition, accompanied by a rapid increase in nascent transcriptional elongation rates. Super elongation complex inhibition restored pausing and suppressed fast-responding transition-associated target genes. In a human cardiac organoid model, inhibition of the super elongation complex prevented EndMT-induced fibrillar collagen deposition and prevented the loss of beating rate. In a hyperlipidemic Pcsk9 gain-of-function mouse model, super elongation complex inhibition administered both prophylactically and therapeutically after established atherosclerosis reduced plaque burden and reduced features of plaque vulnerability. Finally, analysis of 1048 human plaque segments from the Athero-Express biobank showed significant associations between the elongation axis and multiple vulnerability-related plaque traits. Together, these findings identify rapid transcriptional elongation as a mechanistic driver of endothelial plasticity and features of plaque vulnerability and support targeting the elongation machinery as a potential strategy to reduce features of plaque vulnerability in atherosclerotic disease.

Humans↗
PubMed1976

Mechanism of DNA elongation catalyzed by Escherichia coli DNA polymerase III, dnaZ protein, and DNA elongation factors I and III.

Elongation of a primed single-stranded DNA template catalyzed by E. coli DNA polymerase III (DNA nucleotidyltransferase, deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) requires dnaZ protein and two other protein factors, DNA elongation factors I and III. The reaction occurs by the following mechanism: (i) dnaZ protein and DNA elongation factor III together catalyze the transfer of DNA elongation factor I to a primed DNA template. This transfer reaction requires ATP or dATP in addition to dnaZ protein, DNA elongation factors I and III, and primed template; it does not require DNA polymerase III. (ii) DNA polymerase III binds to the complex of DNA elongation factor I with primed template; it does not bind to primed template which is not complexed with DNA elongation factor I. This binding reaction proceeds in the absence of ATP or dATP as cofactor, dnaZ protein, and DNA elongation factor III and without additional DNA elongation factor I. (iii) The complex of DNA polymerase III, DNA elongation factor I, and primed template catalyzes DNA synthesis upon the addition of dNTPs.

Adenosine Triphosphate↗
PubMed1991

The block to transcription elongation at the minute virus of mice attenuator is regulated by cellular elongation factors.

We have previously reported that both in vivo and in vitro, RNA polymerase II pauses or prematurely terminates transcription at a specific attenuation site located 142 to 147 nucleotides downstream from the P4 promoter of minute virus of mice (MVM). In this report, we show that an in vitro block to transcription elongation in HeLa whole-cell extract occurs at elevated KCl concentrations (0.2 to 1.5 M) but not at the standard KCl concentration (50 mM). Briefly initiated transcription complexes, devoid of dissociated elongation factors by passage through a Sephacryl S-1000 column at 0.3 M KCl, were allowed to elongate the briefly initiated nascent RNA, and a block to transcription elongation at the attenuation site was observed independently of the KCl concentration at the time of elongation. Moreover, the block to elongation was overcome by the addition, during elongation, to the column of purified complexes of whole-cell extract from EA cells but not from MVM-infected EA cells or HeLa cells. The general transcription factors IIF and IIX were also shown to alleviate this block to transcription elongation. On the basis of these results, we suggest that the block to elongation at the MVM attenuation site observed late in MVM infection results, at least in part, from the inactivation of the general transcription elongation factors.

Animals↗
PubMed1994

Association of tissue-specific changes in translation elongation after cyclosporin with changes in elongation factor 2 phosphorylation.

In studies of cyclosporin (CsA) toxicity in Sprague-Dawley rats, CsA administered in vivo produced tissue-specific, dose-dependent changes in microsomal translation throughout the bodies of the animals. The most pronounced translation inhibition was in microsomes from the kidney, the organ in which dose-limiting CsA toxicity occurs. In contrast, translation was stimulated in microsomes from the liver. CsA produced changes at the level of translation elongation, which is regulated by the reversible phosphorylation of elongation factor 2 (EF2). Changes in translation elongation after CsA were found to be associated with, and most likely caused by, changes in EF2 phosphorylation. Reduced renal translation elongation was associated with increased EF2 phosphorylation, and increased hepatic elongation with decreased EF2 phosphorylation. EF2 is phosphorylated by Ca2+ calmodulin-dependent protein kinase III (PKIII). Phosphorylated EF2 is a substrate for protein phosphatase 2A (PP2A), but not calcineurin (protein phosphatase 2B or PP2B), the enzyme inhibited by CsA-cyclophilin complexes in T-cells. When CsA or inhibitors of PKIII (EGTA, trifluoperazine) were added in vitro to assays of EF2 phosphorylation in renal or hepatic cytoplasm, or to assays of renal or hepatic microsomal translation elongation, they were without significant effects. Addition in vitro of the PP2A inhibitor okadaic acid increased EF2 phosphorylation in renal and hepatic cytoplasms, but inconsistently produced an inhibition of microsomal translation. However, in less complex rabbit reticulocyte lysates, addition of okadaic acid inhibited PP2A, increased EF2 phosphorylation, and inhibited translation elongation. Furthermore, addition of EGTA and trifluoperazine to rabbit reticulocyte lysates inhibited Ca2+ calmodulin-dependent PKIII activity, decreased EF2 phosphorylation, and stimulated translation elongation. CsA acting alone or as a complex with cyclophilin could alter EF2 phosphorylation by affecting transcriptional regulation or the enzymatic activity of PKIII, PP2A or EF2. Changes in EF2 phosphorylation and translation in body tissues suggest that CsA causes widespread disturbances in phosphorylation and dephosphorylation pathways regulating cellular processes including transcription and translation factor activity. These disturbances may underlie the broad spectrum of toxicities observed during CsA therapy.

Animals↗
PubMed1986

Induction of rat liver mitochondrial fatty acid elongation by the administration of peroxisome proliferator di-(2-ethylhexyl)phthalate: absence of elongation activity in peroxisomes.

The administration of di-(2-ethylhexyl)phthalate (DEHP)3 to male Sprague-Dawley rats resulted in more than a threefold increase in activity of acetyl CoA-dependent hepatic mitochondrial fatty acid elongation. Peroxisomes obtained either from control or DEHP-treated rats were not capable of elongating any of the fatty acyl CoAs tested. Furthermore, the peroxisomes possessed no trans-2-enoyl CoA reductase activity. Therefore, the elongation activity in the 7500g fraction from both control and DEHP-fed animals can be attributed totally to the mitochondria. Maximal incorporation of acetyl CoA occurred in the presence of both NADH and NADPH, and octanoyl CoA (8:0) and decanoyl CoA (10:0) were found to be optimal primers for fatty acid elongation in both control and DEHP-treated animals. The apparent Km for 8:0 CoA was 17 microM in both animal groups while the Vmax was increased from 4.5 to 12.5 nmol/min/mg following treatment. The apparent Km for 10:0 CoA was 10 microM in both control and DEHP-treated groups while the apparent Vmax increased from 2.5 to 10 nmol/min/mg; palmitoyl-CoA (16:0) was a very poor primer for chain elongation. Although the acetyl CoA-dependent fatty acid elongation was stimulated by DEHP treatment, the mitochondrial trans-2-enoyl CoA reductase activity was unaffected. The mitochondrial total elongation activity following DEHP-treatment using 8:0 CoA as primer was about two times higher than enoyl CoA reductase activity using trans-2-decenoyl CoA (10:1). This was the result of accumulation of intermediates, which were identified as trans-2-10:1 (35%), beta-hydroxy 10:0 (25%), unidentified (15%), and elongated saturated product 10:0 (24%). Elongation by one acetate unit was found in both the control and DEHP-treated animals. The results are discussed in terms of physiological significance.

Acetyl Coenzyme A↗
PubMed1985

Regulation of palmitoyl-CoA chain elongation and linoleoyl-CoA chain elongation in rat liver microsomes and the differential effects of peroxisome proliferators, insulin and thyroid hormone.

Treatment of rats with p-chlorophenoxyisobutyric acid (clofibric acid), 2,2'-(decamethylenedithio)diethanol, di(2-ethylhexyl)phthalate or acetylsalicylic acid caused an increase in activity of palmitoyl-CoA chain elongation in hepatic microsomes. The activity of palmitoyl-CoA chain elongation decreased in both hypothyroid-state and diabetic-state rats, increased in hyperthyroid-state rats and did not change in adrenalectomized rats. The administration of clofibric acid to these rats in an altered hormonal state caused an increase in the activity of palmitoyl-CoA chain elongation, but no additional increase in the activity was observed with treatment of hyperthyroid rats with clofibric acid. The activity of linoleoyl-CoA chain elongation did not respond to the changes in either the nutritional conditions or the hormonal state of insulin so sensitively as the activity of palmitoyl-CoA chain elongation. The treatment of rats with triiodothyronine caused a marked increase in the activity of linoleoyl-CoA chain elongation; nevertheless, the activity of linoleoyl-CoA chain elongation was not changed by the treatment of rats with clofibric acid. The results suggest that rat liver microsomes contain at least two fatty acid chain elongation systems and that these chain elongation systems are regulated differently by hormones and drugs.

Acyl Coenzyme A↗
PubMed1995

Effects of the antiterminator BoxA on transcription elongation kinetics and ppGpp inhibition of transcription elongation in Escherichia coli.

It has been shown previously that two different mRNA chains (lacZ and infB) are elongated at a rate of approximately 40 nucleotides (nt)/s during steady state growth on minimal medium and that the rate of mRNA chain elongation is inhibited by ppGpp in vivo. On the other hand, it was found that a truncated ribosomal RNA chain was elongated at a rate of approximately 80 nt/s, independent of growth condition (Vogel, U., and Jensen, K. F. (1994) J. Biol. Chem. 269, 16236-16241). We reasoned that the different transcriptional behavior of mRNA genes and rRNA operons might be caused by the antiterminator sequences present in the rRNA operons. To test this possibility, we have (a) inserted the minimal antiterminator boxA sequence between the promoter and the lacZ and infB genes and (b) deleted the antiterminator sequences from the rRNA transcription unit and measured transcription elongation rates in vivo on the resulting hybrid genes. We found that insertion of boxA in front of the coding region of lacZ increased the transcription elongation rate from 42 nt/s to 69 nt/s during steady state growth and that it eliminated the ppGpp-dependent decrease in the transcription elongation rate during the stringent response. On the other hand, deletion of the antiterminator sequences from the rRNA operon resulted in a reduced transcription elongation rate, but the elongation rate was still insensitive to changes in the ppGpp pool. These results are consistent with the hypothesis that the antiterminator boxA is a primary determinant of the rate of transcription elongation rate.

Base Sequence↗
PubMed1976

Nervonic acid biosynthesis by erucyl-CoA elongation in normal and quaking mouse brain microsomes. Elongation of other unsaturated fatty acyl-CoAs (mono and poly-unsaturated).

Biosynthesis of nervonic acid by enzymatic elongation of erucyl-CoA has been studied in mouse brain microsomes. The substrate and cofactor requirements have been measured. Malonyl-CoA and reduced nicotine-adenine-dinucleotide phosphate are required, but not FMN, FAD or NADH. The effect of protein concentration, incubation time, ATP and CoA has been determined; the reaction products were checked by gas-liquid chromatography with automatic counting of the eluate. Very little activity was found in hydroxylated fatty acids. In the presence of phosphotransacetylase (which impedes the de novo microsomal system), the main reaction product was nervonic acid. It is concluded that nervonic acid is biosynthesised by elongation using a two-carbon unit from malonyl-CoA. The same enzyme biosynthesises saturated and mono-unsaturated very long chain fatty acids. The elongation capacity of "quaking" microsomes is reduced to 30% of the normal value with both erucyl-CoA and behenyl-CoA. Elongation of trans isomer (brassidyl-CoA) and poly-unsaturated homologue (clupanodonyl-CoA) was compared to elongation of erucyl-CoA in both normal and mutant mice. Both unsaturated acyl-CoAs are elongated under the same conditions as erucyl-CoA in brain: the poly-unsaturated acyl-CoA is elongated more actively than the mono-unsaturated acyl-CoA in the mutant.

Animals↗
PubMed1992

The RNA polymerase II elongation complex. Factor-dependent transcription elongation involves nascent RNA cleavage.

Regulation of transcription elongation is an important mechanism in controlling eukaryotic gene expression. SII is an RNA polymerase II-binding protein that stimulates transcription elongation and also activates nascent transcript cleavage by RNA polymerase II in elongation complexes in vitro (Reines, D. (1992) J. Biol. Chem. 267, 3795-3800). Here we show that SII-dependent in vitro transcription through an arrest site in a human gene is preceded by nascent transcript cleavage. RNA cleavage appeared to be an obligatory step in the SII activation process. Recombinant SII activated cleavage while a truncated derivative lacking polymerase binding activity did not. Cleavage was not restricted to an elongation complex arrested at this particular site, showing that nascent RNA hydrolysis is a general property of RNA polymerase II elongation complexes. These data support a model whereby SII stimulates elongation via a ribonuclease activity of the elongation complex.

Animals↗
PubMed1991

RNA polymerase II elongation complex. Elongation complexes purified using an anti-RNA antibody do not contain initiation factor alpha.

Gene expression in eukaryotes can be regulated by controlling the efficiency of transcript elongation by RNA polymerase II. The composition of the elongation complex is, however, poorly understood. Previous work has identified DNA sequences which block RNA polymerase II transcription and factors which stimulate RNA chain elongation. Here, I have purified elongation complexes arrested at discrete template locations. Complexes were rapidly and efficiently precipitated from in vitro transcription reactions using a monoclonal antibody that binds RNA. The isolated complexes remained transcriptionally active. This technique enables the facile manipulation of transcription elongation complexes. Using this approach, I show that transcription initiation factor alpha is not associated with a RNA polymerase II elongation complex. Since others have shown that alpha associates stoichiometrically with DNA, RNA polymerase II, and other required factors in an initiation complex, this work suggests that alpha departs from the transcription complex after nucleotides are required but before extensive RNA chain synthesis. In this regard alpha resembles the bacterial promoter-recognition factor sigma.

Animals↗
PubMed1994

RNA chain elongation and termination by mammalian RNA polymerase III. Analysis of tRNA gene transcription by imposing a reversible factor-mediated block to elongation using a sequence-specific DNA binding protein.

We have used a sequence-specific DNA binding protein to examine transcription elongation and termination by mammalian RNA polymerase III (polIII). The Escherichia coli lac repressor protein, bound to its cognate operator site positioned between the 3' end of the coding region and the termination site of a human tRNA gene, conditionally blocked transcription elongation by polIII in vitro in HeLa cell nuclear extracts. Arrest of elongation by polIII dramatically reduced overall levels of transcription and directed the synthesis of shortened transcripts, consistent with a block to polIII elongation at the boundary of the repressor/DNA complex. Removal of template-bound repressor with the allosteric inducer isopropylthio-beta-D-galactoside (IPTG) allowed extension of nascent transcripts and restored transcriptional activity. Moreover, a subset of transcription complexes were shown to be capable of transcribing through the repressor obstacle. lac repressor positioned just downstream of the natural termination site effected the premature termination of transcription but otherwise had no affect on the overall level of transcription. Our findings demonstrate that elongation and termination by mammalian polIII can be modulated in vitro by a heterologous sequence-specific DNA binding protein. Moreover, the ability to selectivity arrest elongation by polIII at defined positions within the tRNA gene transcription unit has permitted the identification of discrete functional properties of paused mammalian polIII ternary complexes.

Base Sequence↗
PubMed1995

Transgene-mediated auxin overproduction in Arabidopsis: hypocotyl elongation phenotype and interactions with the hy6-1 hypocotyl elongation and axr1 auxin-resistant mutants.

Transgenic Arabidopsis thaliana plants constitutively expressing Agrobacterium tumefaciens tryptophan monooxygenase (iaaM) were obtained and characterized. Arabidopsis plants expressing iaaM have up to 4-fold higher levels of free indole-3-acetic acid (IAA) and display increased hypocotyl elongation in the light. This result clearly demonstrates that excess endogenous auxin can promote cell elongation in a whole plant. Interactions of the auxin-overproducing transgenic plants with the phytochrome-deficient hy6-1 and auxin-resistant axr1-3 mutations were also studied. The effects of auxin overproduction on hypocotyl elongation were not additive to the effects of phytochrome deficiency in the hy6-1 mutant, indicating that excess auxin does not counteract factors that limit hypocotyl elongation in hy6-1 seedlings. Auxin-overproducing seedlings are also qualitatively indistinguishable from wild-type controls in their response to red, far-red, and blue light treatments, demonstrating that the effect of excess auxin on hypocotyl elongation is independent of red and blue light-mediated effects. All phenotypic effects of iaaM-mediated auxin overproduction (i.e. increased hypocotyl elongation in the light, severe rosette leaf epinasty, and increased apical dominance) are suppressed by the auxin-resistant axr1-3 mutation. The axr1-3 mutation apparently blocks auxin signal transduction since it does not reduce auxin levels when combined with the auxin-overproducing transgene.

Arabidopsis↗