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At least 235 records · Page 13Linked to original sources
PubMed2006

The E7 oncoprotein is translated from spliced E6*I transcripts in high-risk human papillomavirus type 16- or type 18-positive cervical cancer cell lines via translation reinitiation.

High-risk human papillomaviruses (HPVs) encode two viral oncoproteins, E6 and E7, from a single bicistronic pre-mRNA containing three exons and two introns. Retention of intron 1 in the E6 coding region is essential for production of the full-length E6 oncoprotein. However, splicing of intron 1 is extremely efficient in cervical cancer cells, leading to the production of a spliced transcript, E6*I, of E6. Here, we investigated whether this splicing of intron 1 might benefit E7 production. Using RNA interference as a tool, we targeted the intron 1 region using small interfering RNAs (siRNAs) in HPV-positive cell lines. At an effective low dose, the siRNAs specifically suppressed E6 expression but not E7 expression, as demonstrated by the stabilization of p53. However, at high doses the HPV18 intron 1-specific siRNA substantially and specifically reduced the level of the 18E6*I mRNA lacking the intron region in HeLa cells, implying its nuclear silencing on the pre-mRNA before RNA splicing. Two other siRNAs targeting the exon 2 regions of HPV16 and -18, which encode the E7 oncoprotein, reduced the E6*I mRNAs to a remarkable extent and preferentially suppressed expression of E7, leading to accumulation of hypophosphorylated p105Rb and cell cycle arrest, indicating that the majority of E7 proteins are the translational products of E6*I mRNAs. This was confirmed by transient transfection in 293 cells: E7 could be translated only from the E7 open reading frame (ORF) on E6*I mRNA in a distance-dependent matter of upstream E6*I ORF by translation reinitiation. The data thus provide direct evidence that the E6*I mRNAs of high-risk HPVs are responsible for E7 production.

Cell Line↗
PubMed2001

Self-initiation of translation of mRNAs devoid of translational initiators in Escherichia coli.

Recent studies have shown that the canonical SD-anti-SD interaction is dispensable for the initiation of translation of certain mRNAs in Escherichia coli. In this study the cat and tetR genes were modified to either destroy complementarity to E. coli 16S RNA or completely delete their 5' non-translated regions. Thus a series of cat- and tetR-derived genes were constructed, cloned under a strong constitutive promoter and expressed in E. coli cells. The efficiency of expression was evaluated by the yield of CAT (for the cat gene) and cell viability in increasing concentrations of antibiotic (for both cat and tetR genes). The obtained results show that the mRNAs transcribed from both series of reporter genes (cat and tetR) were active in vivo. Their activity was preserved even in the cases when the length of their 5' non-translated leader sequences was reduced to one nucleotide for the cat gene and eight nucleotides for the tetR gene. The yield of protein obtained with the latter constructs was detectable and sufficient for bacteria to survive at 50-100 microg/ml chloramphenicol and 20 microg/ml tetracycline, respectively.

5' Untranslated Regions↗
PubMed1992

Macrophage and monocyte IL-1 beta regulation differs at multiple sites. Messenger RNA expression, translation, and post-translational processing.

Maturation of blood monocytes into macrophages is accompanied by a number of functional changes including decreased IL-1 beta release in response to LPS. This limitation has previously been ascribed to transcriptional regulation. However, in seeming conflict with the observed depression in IL-1 beta mRNA levels, recent work demonstrates increased intracellular IL-1 beta in macrophages. Therefore, the present study sought to explain these differences by comparing IL-1 beta production from autologous alveolar macrophage and blood monocyte pairs at multiple regulatory sites, including endotoxin responsiveness, mRNA expression, protein translation, and post-translational processing. Macrophages did not differ from monocytes in endotoxin sensitivity, but when analyzed by both ELISA and Western blot, were confirmed to have limitations in IL-1 beta release. Gene expression studies demonstrated that at 4 h, macrophage IL-1 beta steady state mRNA levels were 3-fold lower than the monocyte's. However, total IL-1 beta protein production, as measured by [35S]methionine labeling with immunoprecipitation, demonstrated three- to sixfold higher amounts in macrophages at comparable time points. The enhanced protein production in the face of relatively low mRNA levels suggests that macrophages translate IL-1 beta mRNA more efficiently. Furthermore, characterization of IL-1 beta release into supernatants revealed that whereas monocyte release occurred early, represented 5 to 20% of the intracellular amounts, and contained largely processed IL-1 beta, macrophage release was delayed, represented 1 to 5% of the intracellular amounts, and contained primarily unprocessed IL-1 beta. Taken together, these data demonstrate that the limitations in alveolar macrophage IL-1 beta release occur due to slower export and conversion of 35- to 17-kDa protein and are not due to differences in sensitivity to endotoxin or to transcriptional control mechanisms.

Blotting, Northern↗
PubMed1992

FAD-dependent regulation of transcription, translation, post-translational processing, and post-processing stability of various mitochondrial acyl-CoA dehydrogenases and of electron transfer flavoprotein and the site of holoenzyme formation.

The most prominent biochemical consequence of riboflavin deficiency in rats is a drastic decrease in various acyl-CoA dehydrogenase activities, especially that of short chain and isovaleryl-CoA dehydrogenase (IVD). As a result, oxidation of fatty acids and leucine is severely inhibited. We studied the effects of FAD at various stages of acyl-CoA dehydrogenase biogenesis. Immunoblot revealed severe losses of various acyl-CoA dehydrogenases and electron transfer flavoprotein in riboflavin-deficient rat liver mitochondria. The decreases in IVD and short chain acyl-CoA dehydrogenase were particularly severe, reaching values of 17 and 34% of controls, respectively. With the exception of IVD, the rate of in vitro transcription of the respective genes and the amounts of mRNAs of these flavoproteins in tissues increased 3-8.5-fold over controls. The amount of IVD mRNA and its transcription rate remained unchanged, suggesting that IVD expression is regulated separately from other acyl-CoA dehydrogenases. When riboflavin was depleted, in vitro translation of acyl-CoA dehydrogenase and electron transfer flavoprotein alpha-subunit mRNAs was moderately inhibited. Translation of non-flavoproteins was also inhibited. The stability of precursor acyl-CoA dehydrogenases and their mitochondrial import/processing were unaffected. However, mature acyl-CoA dehydrogenases degraded markedly faster in deficient mitochondria than in controls. Regardless of whether precursors were translated under riboflavin-depleted or riboflavin replete conditions, mature acyl-CoA dehydrogenases survived well when imported into normal mitochondria but degraded faster when imported into deficient mitochondria. These findings indicate that FAD ligand binds to mature acyl-CoA dehydrogenase inside the mitochondria.

Acyl-CoA Dehydrogenases↗
PubMed2005

Translating a single-word items scale with multiple subcomponents--A Hebrew translation of the Profile of Mood States.

BACKGROUND: The purpose of the study was to assess the translation adequacy of a Hebrew version of the POMS, and to report the decision-making process of overcoming methodological flaws. METHOD: The English version was translated to Hebrew by expert team approach. Factor analysis for construct validation was predominantly used for assessment, in addition to other techniques: back translation, use of bilingual subjects, and correlation with other instruments for concurrent validation. RESULTS: The final 28-item Hebrew version has suitable psychometric properties. Of the six dimensions on the English version scale, five dimensions--"anger," "depression:' "fatigue," "tension" and "vigor"--were found comparable to the English version, while "confusion" was deleted. LIMITATIONS: The pre-testing was performed on students only. CONCLUSIONS: Although the study shows valid psychometric properties, further studies in various populations are needed to assess the sensitivity of the scale to diverse manipulations.

Adult↗
PubMed1986

Demonstration of post-translational secretion of human placental lactogen by a mammalian in vitro translation system.

This study demonstrates the post-translational translocation across the rough endoplasmic reticular membrane of a mammalian secretory protein, human preplacental lactogen. In the rabbit reticulocyte lysate, human preplacental lactogen biosynthesis is arrested by addition of cycloheximide prior to supplementation with dog pancreatic microsomal membranes, which have previously been shown to translocate and process nascent secretory proteins in a cotranslational manner. Twenty-five percent of the precursor protein is consistently converted to its mature form under these post-translational conditions. The resulting mature hormone is resistant to proteolytic degradation by added proteases, thus indicating that it is translocated across the microsomal membrane and sequestered within the lumenal space of the microsomal vesicles. Approximately one-half of the precursor protein synthesized is associated with the ribosomes. Only the ribosome-associated fraction is secreted in this in vitro system, suggesting that the process of post-translational secretion requires ribosomes for protein interaction with the elements of a subcellular secretory apparatus.

Animals↗
PubMed2006

Tolerance for random recombination of domains in prokaryotic and eukaryotic translation systems: Limited interdomain misfolding in a eukaryotic translation system.

It has been proposed that eukaryotic translation systems have a greater capacity for cotranslational folding of domains than prokaryotic translation systems, which reduces interdomain misfolding in multidomain proteins and, therefore, leads to tolerance for random recombination of domains. However, there has been a controversy as to whether prokaryotic and eukaryotic translation systems differ in the capacity for cotranslational domain folding. Here, to examine whether these systems differ in the tolerance for the random domain recombination, we systematically combined six proteins, out of which four are soluble and two are insoluble when produced in an Escherichia coli and a wheat germ cell-free protein synthesis systems, to construct a fusion protein library. Forty out of 60 two-domain proteins and 114 out of 120 three-domain proteins were more soluble when produced in the wheat system than in the E. coli system. Statistical analyses of the solubilities and the activities indicated that, in the wheat system but not in the E. coli system, the two soluble domains comprised mainly of beta-sheets tend to avoid interdomain misfolding and to fold properly even at the neighbor of the misfolded domains. These results demonstrate that a eukaryotic system permits the concomitance of a wider variety of domains within a single polypeptide chain than a prokaryotic system, which is probably due to the difference in the capacity for cotranslational folding. This difference is likely to be related to the postulated difference in the tolerance for random recombination of domains.

Escherichia coli↗
PubMed1995

Synthesis of non-translating or translating specialized ribosomes causes feedback regulation of ribosomal RNA synthesis in Escherichia coli.

Specialized ribosomes carry a mutant anti-Shine-Dalgarno region that disrupts the complementary base pairing that stabilizes the translation initiation complex with E. coli mRNAs. It has been reported that production of specialized ribosomes does not cause the inhibition of chromosomal rRNA synthesis that follows production of wild-type ribosomes. We proposed that enabling translation on specialized ribosomes by providing mRNA with a complementary mutation in the Shine-Dalgarno region would restore feedback regulation and inhibit chromosomal rRNA synthesis. With both our system and the system studied previously, we saw feedback regulation regardless of whether the specialized ribosomes were translating. As reported previously, transcription from plasmid-borne promoters decreased as chromosomal rRNA synthesis was repressed, suggesting that the lambda PL and tac promoters may be sensitive to the effector(s) of feedback regulation.

Base Sequence↗
PubMed1997

Translational activation in coliphage Qbeta: on a polycistronic messenger RNA, repression of one gene can activate translation of another.

We present evidence for translational activation of the Qbeta coliphage maturation cistron, mediated by the presence of Qbeta replicase. This activation does not require RNA replication, translation of a second gene, or any direct protein-RNA binding at the maturation gene initiation site. Our data support a model in which the Qbeta maturation gene remains translationally "off" by two means: (1) the thermodynamic stability of an RNA structure that greatly discourages, but does not eliminate, ribosome access at the maturation start site; and (2) the presence of the stronger, proximal coat gene ribosome binding site. Moreover, maturation gene expression is switched "on" when ribosome entry at the coat initiation site, present on the same polycistronic RNA molecule, is repressed by Qbeta replicase, thereby allowing ribosomes to compete for the weaker, upstream maturation start site.

Allolevivirus↗
PubMed2001

The RING domains of the promyelocytic leukemia protein PML and the arenaviral protein Z repress translation by directly inhibiting translation initiation factor eIF4E.

The promyelocytic leukemia protein (PML) is a mammalian regulator of cell growth which is characteristically disrupted in acute promyelocytic leukemia and by a variety of viruses. PML contains a RING domain which is required for its growth-suppressive and antiviral properties. Although normally nuclear, in certain pathogenic conditions, including arenaviral infection, PML is relocated to the cytoplasm, where its functions are poorly understood. Here, we observe that PML and arenavirus protein Z use regions around the first zinc-binding site of their respective RING domains to directly interact, with sub-micromolar affinity, with the dorsal surface of translation initiation factor eIF4E, representing a novel mode of eIF4E recognition. PML and Z profoundly reduce the affinity of eIF4E for its substrate, the 5' 7-methyl guanosine cap of mRNA, by over 100-fold. Association with the dorsal surface of eIF4E and direct antagonism of mRNA cap binding by PML and Z lead to direct inhibition of translation. These activities of the RING domains of PML and Z do not involve ubiquitin-mediated protein degradation, in contrast to many RINGs which have been observed to do so. Although PML and Z have well characterized physiological functions in regulation of growth and apoptosis, this work establishes the first discrete biochemical mechanism which underlies the biological activities of their RING domains. Thus, we establish PML and Z as translational repressors, with potential contributions to the pathogenesis of acute promyelocytic leukemia and variety of viral infections.

Arenavirus↗
PubMed1990

In-vitro translation of cucumoviral satellites. III. Translational efficiencies of cucumber mosaic virus-associated RNA 5 sequence variants can be related to the predicted secondary structures of their first 55 nucleotides.

The cucumber mosaic virus (CMV) satellites D- and S-CARNA 5 (CARNA 5 = Cucumber mosaic virus-Associated RNA 5), their full-length cDNA clone transcripts, and DNA clone transcripts of their open reading frames (ORFs) were used as mRNAs in the wheat-germ in-vitro translation system. Natural D-CARNA 5 yielded an anomalously large polypeptide, while transcripts made from cDNA clones of D-CARNA 5 or its first ORF had no mRNA activity. Transcripts made from the second major ORF in D-CARNA 5 yielded a smaller product, consistent with its size. Natural S-CARNA 5 and its cDNA clone transcripts both yielded the two polypeptides previously reported, while transcripts of its only major ORF yielded exclusively the smaller of the two products. The potential for an alternate initiation codon, 36 nucleotides upstream, being the source of the larger of the two polypeptides was tested. The differences in the translational properties of D- and S-CARNA 5 were related to the predicted secondary structures of the first 55 nucleotides in these CARNA 5 sequence variants. The calculated free energies of the predicted hairpins correlated inversely with their in-vitro translational activities.

Base Sequence↗
PubMed1983

The translation of the messenger for the poly(A)-binding protein-associated with translated mRNA is suppressed. A case of cytoplasmic repression in duck erythroblasts.

In vivo protein synthesis in duck erythroblasts was compared to in vitro translation of polyribosomal and free cytoplasmic mRNA. The in vivo study showed the absence of de novo synthesis of the Mr 73 000 poly(A)-binding protein found associated with all polyribosomal mRNA. In vitro translation demonstrated that the mRNA for this protein is absent from the polyribosomal mRNA fraction but constitutes a medium frequency messenger among the repressed free mRNA. This result confirms the existence of a qualitative translational control in terminal differentiating duck erythroblasts leading eventually to the arrest of the protein synthesizing machinery.

Animals↗
PubMed1990

A nucleotide sequence in the translation start signal region is involved in heat shock-induced translation arrest in Escherichia coli.

In Escherichia coli synthesis of several proteins is transiently depressed upon heat shock treatment. A comparison of nucleotide sequences of the genes encoding these proteins revealed the occurrence of a consensus sequence, GAGGAA(N)3-6ATG, in their translation start signal region. To examine whether this sequence is involved in heat shock-induced depression of protein synthesis, DNA segments corresponding to this region of four of these genes, fusA, rpoB, glnS, and pheT, were synthesized, and each of them was fused in frame with the lacZ gene on the open reading frame vector pORF1. The effect of heat shock on the synthesis of beta-galactosidase encoded by these fused genes was then studied in E. coli. It was thus found that beta-galactosidase synthesis starting from the inserted translation start signal was arrested transiently upon temperature shift-up from 30 to 42 degrees C. I conclude that the heat shock-induced depression of gene expression is an event taking place at the initiation of translation.

Base Sequence↗
PubMed1992

Both forms of translational initiation factor IF2 (alpha and beta) are required for maximal growth of Escherichia coli. Evidence for two translational initiation codons for IF2 beta.

The gene infB codes for two forms of translational initiation factor IF2; IF2 alpha (97,300 Da) and IF2 beta (79,700 Da). IF2 beta arises from an independent translational event on a GUG codon located 471 bases downstream from IF2 alpha start codon. By site-directed mutagenesis we constructed six different mutations of this GUG codon. In all cases, IF2 beta synthesis was variably affected by the mutations but not abolished. We show that the residual expression of IF2 beta results from translational initiation on an AUG codon located 21 bases downstream from the mutated GUG. Furthermore, two forms of IF2 beta have been separated by fast protein liquid chromatography and the determination of their N-terminal sequences indicated that they resulted from two internal initiation events, one occurring on the previously identified GUG start codon, the other on the AUG codon immediately downstream. We conclude that two forms of IF2 beta exist in the cell, which differ by seven aminoacid residues at their N terminus. Only by mutating both IF2 beta start codons could we construct plasmids that express only IF2 alpha. A plasmid expressing only IF2 beta was obtained by deletion of the proximal region of the infB gene. Using a strain that carries a null mutation in the chromosomal copy of infB and a functional copy of the same gene on a thermosensitive lysogenic lambda phage, we could cure the lambda phage when the plasmids expressing only one form of IF2 were supplied in trans. We found that each one of the two forms of IF2, at near physiological levels, can support growth of Escherichia coli, but that growth is retarded at 37 degrees C. This result shows that both forms of IF2 are required for maximal growth of the cell and suggests that they have acquired some specialized but not essential function.

Amino Acid Sequence↗
PubMed2005

The nanos translational control element represses translation in somatic cells by a Bearded box-like motif.

Developmental control of translation is frequently mediated by regulatory elements that reside within 3' untranslated regions (3' UTRs). Two stem-loops within the nanos 3' UTR translational control element (TCE) act independently to direct translational repression of maternal nanos mRNA in the ovary or embryo. We have previously shown that the nanos TCE can also function in select somatic sites. Using an ectopic expression screen, we now identify a new site of TCE function, the dorsal pouch epithelium. Analysis of TCE mutants reveals that TCE activity in the dorsal pouch does not depend on either of the stem-loops required for maternal TCE function, but instead requires a third feature-a sequence that closely matches the Bearded box, a regulatory motif found in the 3' UTRs of several Notch pathway genes. In addition, we identify pleiohomeotic mRNA as an endogenous candidate for regulation by Bearded box-like motifs in the dorsal pouch. Together, these results suggest that the TCE has appropriated a conserved regulatory motif to expand its function to somatic tissues.

3' Untranslated Regions↗
PubMed2000

Translational frameshifting: implications for the mechanism of translational frame maintenance.

The ribosome rapidly translates the information in the nucleic sequence of mRNA into the amino acid sequence of proteins. As with any biological process, translation is not completely accurate; it must compromise the antagonistic demands of increased speed and greater accuracy. Yet, reading-frame errors are especially infrequent, occurring at least 10 times less frequently than other errors. How do ribosomes maintain the reading frame so faithfully? Geneticists have addressed this question by identifying suppressors that increase error frequency. Most familiar are the frameshift suppressor tRNAs, though other suppressors include mutant forms of rRNA, ribosomal proteins, or translation factors. Certain mRNA sequences can also program frameshifting by normal ribosomes. The models of suppression and programmed frameshifting describe apparently quite different mechanisms. Contemporary work has questioned the long-accepted model for frameshift suppression by mutant tRNAs, and a unified explanation has been proposed for both phenomena. The Quadruplet Translocation Model proposes that suppressor tRNAs cause frameshifting by recognizing an expanded mRNA codon. The new data are inconsistent with this model for some tRNAs, implying the model may be invalid for all. A new model for frameshift suppression involves slippage caused by a weak, near-cognate codon.anticodon interaction. This strongly resembles the mechanism of +1 programmed frameshifting. This may mean that infrequent frameshift errors by normal ribosomes may result from two successive errors: misreading by a near-cognate tRNA, which causes a subsequent shift in reading frame. Ribosomes may avoid phenotypically serious frame errors by restricting apparently innocuous errors of sense.

Anticodon↗
PubMed2000

Isolation and functional characterization of a temperature-sensitive mutant of the yeast Saccharomyces cerevisiae in translation initiation factor eIF5: an eIF5-dependent cell-free translation system.

Eukaryotic translation initiation factor 5 (eIF5) interacts with the 40S ribosomal initiation complex (40S.eIF3.AUG.Met-tRNA(f).eIF2.GTP) to promote the hydrolysis of bound GTP. In Saccharomyces cerevisiae, eIF5, a protein of 45346Da, is encoded by a single-copy essential gene, TIF5. In this paper, we have isolated a temperature-sensitive S. cerevisiae strain, TMY5-1, by replacing the wild-type chromosomal copy of TIF5 with one mutagenized in vitro. The mutant yeast cells rapidly cease protein synthesis when grown under non-permissive conditions, lose polyribosomes and accumulate free 80S ribosomes. Further characterization of mutant eIF5 showed that the mutant protein, expressed in Escherichia coli, is defective both in its interaction with eIF2 as well as in mediating the hydrolysis of GTP bound to the 40S initiation complex and consequently in the formation of the 80S initiation complex. Additionally, the availability of a yeast strain containing temperature-sensitive mutation in the eIF5 gene allowed us to construct a cell-free translation system that was dependent on exogenously added eIF5 for translation of mRNAs in vitro.

Cell-Free System↗