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At least 163 records · Page 9Linked to original sources

PubMed2000

Defects in tRNA processing and nuclear export induce GCN4 translation independently of phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2.

Induction of GCN4 translation in amino acid-starved cells involves the inhibition of initiator tRNA(Met) binding to eukaryotic translation initiation factor 2 (eIF2) in response to eIF2 phosphorylation by protein kinase GCN2. It was shown previously that GCN4 translation could be induced independently of GCN2 by overexpressing a mutant tRNA(AAC)(Val) (tRNA(Val*)) or the RNA component of RNase MRP encoded by NME1. Here we show that overexpression of the tRNA pseudouridine 55 synthase encoded by PUS4 also leads to translational derepression of GCN4 (Gcd(-) phenotype) independently of eIF2 phosphorylation. Surprisingly, the Gcd(-) phenotype of high-copy-number PUS4 (hcPUS4) did not require PUS4 enzymatic activity, and several lines of evidence indicate that PUS4 overexpression did not diminish functional initiator tRNA(Met) levels. The presence of hcPUS4 or hcNME1 led to the accumulation of certain tRNA precursors, and their Gcd(-) phenotypes were reversed by overexpressing the RNA component of RNase P (RPR1), responsible for 5'-end processing of all tRNAs. Consistently, overexpression of a mutant pre-tRNA(Tyr) that cannot be processed by RNase P had a Gcd(-) phenotype. Interestingly, the Gcd(-) phenotype of hcPUS4 also was reversed by overexpressing LOS1, required for efficient nuclear export of tRNA, and los1Delta cells have a Gcd(-) phenotype. Overproduced PUS4 appears to impede 5'-end processing or export of certain tRNAs in the nucleus in a manner remedied by increased expression of RNase P or LOS1, respectively. The mutant tRNA(Val*) showed nuclear accumulation in otherwise wild-type cells, suggesting a defect in export to the cytoplasm. We propose that yeast contains a nuclear surveillance system that perceives defects in processing or export of tRNA and evokes a reduction in translation initiation at the step of initiator tRNA(Met) binding to the ribosome.

Alcohol Oxidoreductases↗

PubMed2001

Linking the 3' poly(A) tail to the subunit joining step of translation initiation: relations of Pab1p, eukaryotic translation initiation factor 5b (Fun12p), and Ski2p-Slh1p.

The 3' poly(A) structure improves translation of a eukaryotic mRNA by 50-fold in vivo. This enhancement has been suggested to be due to an interaction of the poly(A) binding protein, Pab1p, with eukaryotic translation initiation factor 4G (eIF4G). However, we find that mutation of eIF4G eliminating its interaction with Pab1p does not diminish the preference for poly(A)(+) mRNA in vivo, indicating another role for poly(A). We show that either the absence of Fun12p (eIF5B), or a defect in eIF5, proteins involved in 60S ribosomal subunit joining, specifically reduces the translation of poly(A)(+) mRNA, suggesting that poly(A) may have a role in promoting the joining step. Deletion of two nonessential putative RNA helicases (genes SKI2 and SLH1) makes poly(A) dispensable for translation. However, in the absence of Fun12p, eliminating Ski2p and Slh1p shows little enhancement of expression of non-poly(A) mRNA. This suggests that Ski2p and Slh1p block translation of non-poly(A) mRNA by an effect on Fun12p, possibly by affecting 60S subunit joining.

DEAD-box RNA Helicases↗

PubMed1991

Cytoplasmic localization during storage and translation of the mRNAs of transition protein 1 and protamine 1, two translationally regulated transcripts of the mammalian testis.

During spermatogenesis in mammals, the transcripts of transition protein 1 (TP 1) and protamine 1 (Prm 1) are under translational regulation. Following their transcription in round spermatids, the mRNAs for TP 1 and Prm 1 are stored in the cytoplasm from 3-7 days before being translated towards the end of spermatogenesis. To test the hypothesis that the inactivation or activation of transcripts during spermiogenesis could be mediated by mRNA compartmentalization in the cytoplasm of spermatids, light and electron microscopy were used to localize, by in situ hybridization, the cellular and subcellular sites of stored and translated mRNAs for these two testis-specific transcripts. During early spermiogenesis (before step 7) nuclear transcripts of both TP 1 and Prm 1 were seen. After step 7 the TP 1 and Prm 1 mRNAs were only detected in the cytoplasm. Throughout spermiogenesis the cytoplasmic mRNAs were not localized to any membrane-bound organelles such as the endoplasmic reticulum or mitochondria or to non-membrane-bound structures such as the chromatoid body. These studies demonstrate that the translational arrest of the TP 1 and Prm 1 mRNAs is not primarily controlled by compartmentalized storage in the cytoplasm of spermatids. Moreover, when translation of these mRNAs occurs in elongated spermatids, the mRNAs are present throughout the cytoplasm.

Animals↗

PubMed2006

Sulfolobus solfataricus translation initiation factor 1 stimulates translation initiation complex formation.

The eukaryotic translation initiation factor 1 binds to the ribosome during translation initiation. It is instrumental for initiator-tRNA and mRNA binding, and has a function in selection of the authentic start codon. Here, we show that the archaeal homolog aIF1 has analogous functions. The aIF1 protein of the archaeon Sulfolobus solfataricus is bound to the small ribosomal subunit during translation initiation and accelerates binding of initiator-tRNA and mRNA to the ribosome. Accordingly, aIF1 stimulated translation of an mRNA in a S. solfataricus in vitro translation system. Moreover, this study suggested that the C terminus of the factor is of relevance for its function.

Amino Acid Sequence↗

PubMed2006

mRNA translation is compartmentalized to the endoplasmic reticulum following physiological inhibition of cap-dependent translation.

Eukaryotic cells utilize a cycle of ribosome trafficking on the endoplasmic reticulum (ER) to partition mRNAs between the cytosol and ER compartments. In this process, ribosomes engaged in the synthesis of signal sequence-bearing proteins are trafficked to the endoplasmic reticulum via the signal-recognition particle pathway and are released from the ER upon translation termination. Though the processes governing ribosome trafficking to the ER are well understood, little is known regarding the complementary ribosome release process. In this study, Coxsackie B virus (CBV) infection was used to inactivate the initiation stage of protein synthesis, thereby limiting translation to the elongation and termination stages. Ribosome partitioning between the cytosol and ER compartments was examined to determine the role of termination in ribosome release from the ER. CBV infection resulted in efficient cleavage of eIF4G and PABP, coincident with polyribosome breakdown in the cytosol and ER compartments. Termination resulted in the continued association of ribosomes with the ER compartment, rather than the expected process of ribosome release. Analyses of ribosome/mRNA loading patterns in the cytosol and ER revealed that CBV infection was accompanied by a suppression of mRNA translation in the cytosol and the sustained, although reduced, translation in the ER compartment. Direct biosynthetic labeling experiments demonstrated that protein synthesis on the ER was enhanced relative to the cytosol following CBV infection. In total, these data demonstrate that ribosome and mRNA release from the ER is regulated independent of translation termination and identify the ER as a privileged site for protein synthesis.

Blotting, Northern↗

PubMed2003

Chloride concentration discriminates between Foot-and-mouth disease virus ires-dependent translation and classical scanning translation: new aspects of the picornavirus shutoff mechanism.

Some picornaviruses might use the general increase of ionic strength in the host cell that occurs successively after infection to induce shutoff of host protein synthesis and to stimulate viral protein synthesis. In order to investigate this discrimination mode on a molecular level, in vitro experiments under different salt conditions comparing the Foot-and-mouth disease virus (FMDV) internal ribosome entry site (IRES)-dependent translation with the translation via the classical scanning mechanism were performed. For classical mRNA optimum concentrations of all investigated salts ranged between 70 and 100 mmol/l. However, for FMDV IRES-dependent translation the optima depended strongly on the anion used. While acetates caused only a weak stimulation of translation efficiency with maxima ranging between 150 and 180 mmol/l, chlorides lead to a strong stimulation with maxima ranging between 120 and 150 mmol/l. Competition experiments revealed that the concentration of chlorides had a greater influence on the discrimination between cellular and viral RNA translation than the total ionic strength. Taken together, the data support a model in which a specific increase in the chloride concentration rather than a general increase in the ionic strength is responsible for the shutoff effect induced by some picornaviruses.

Cations, Monovalent↗

PubMed1992

Translational control by influenza virus. Selective and cap-dependent translation of viral mRNAs in infected cells.

In cells infected by influenza virus type A, host protein synthesis undergoes a rapid and dramatic shutoff. To define the molecular mechanisms underlying this selective translation, a transfection/infection protocol was developed utilizing viral and cellular cDNA clones. When COS-1 cells were transfected with cDNAs encoding nonviral genes and subsequently infected with influenza virus, protein expression from the exogenous genes was diminished, similar to the endogenous cellular genes. However, when cells were transfected with a truncated influenza viral nucleocapsid protein (NP-S) gene, the NP-S protein was made as efficiently in influenza virus infected cells as in uninfected cells, showing that the NP-S mRNA, although expressed independently of the influenza virus replication machinery, was still recognized as a viral and not a cellular mRNA. Northern blot analysis demonstrated that the selective blocks to nonviral protein synthesis were at the level of translation. Moreover, polysome experiments revealed that the translational blocks occurred at both the initiation and elongation stages of cellular protein synthesis. Finally, we utilized this transfection/infection system as well as double infection experiments to demonstrate that the translation of influenza viral mRNAs probably occurred in a cap-dependent manner as poliovirus infection inhibited influenza viral mRNA translation.

Capsid↗

PubMed1988

The iron-responsive element is the single element responsible for iron-dependent translational regulation of ferritin biosynthesis. Evidence for function as the binding site for a translational repressor.

Ferritin, a cytoplasmic protein critical in iron metabolism, displays iron-dependent regulation of its biosynthetic rate with no corresponding changes in mRNA levels. An iron-responsive element (IRE) has been identified in the 5'-untranslated region (UTR) of the human ferritin heavy chain mRNA which, when placed in the 5'-UTR of heterologous reporter genes, confers iron-dependent translational regulation to the hybrid mRNAs. However, whereas the biosynthetic rate of ferritin in response to changes in iron status exhibits a 30-80-fold range, the apparent ranges observed for reporter gene constructs utilizing chloramphenicol acetyltransferase assays or human growth hormone radioimmunoassays have been much less. A deletion and reconstitution study was undertaken to address the possibility that regions of the ferritin gene and mRNA other than the IRE may be necessary for the production of the full range of iron regulation. Data are presented that demonstrate that the IRE alone is capable of conferring iron-dependent translational regulation of biosynthesis to downstream encoded proteins that is both qualitatively and quantitatively similar to that observed with expression of ferritin itself. Thus, the complete range of iron-dependent translational regulation conferred by the IRE occurs independently of the presence of the ferritin promoter, other regions of the ferritin 5'-UTR, the ferritin coding region, and the ferritin 3'-UTR. Additionally, experiments addressing the translatability in vivo of various ferritin construct mRNAs support the theory that the IRE functions as the binding site for a translational repressor.

Binding Sites↗

PubMedDate not supplied

[Translation of RNA 4 from plant brome mosaic virus in an Escherichia coli cell-free system with pure protein factors in translation].

Translation by RNA 4 from plant brome mosaic virus coding for the virus coat protein in an E. coli cell-free system with pure factors of translation has been studied. It has been shown that the initiation of translation by this mRNA depends completely on the three E. coli initiation factors. Optimal ionic conditions for the formation of the initiatory 70S times fMet-tRNA times RNA 4 complex have been found. It has been shown that this complex is stable in conditions of zonal centrifugation. On the basis of reaction with puromycin it has been determined that the initiatory fMet-tRNA in this complex occupies the donor-tRNA-binding site of the ribosome. By the competence of the initiatory ribosomal complex for binding with Ser-tRNA (serine is the N-terminal amino acid in the virus coat protein) it can be concluded that the ribosomal and the E. coli initiation factors recognize the initiatory codon of the RNA r from brome mosaic virus. Peptide synthesis induced by RNA 4 has been obtained on E. coli ribosomes with five pure factors of translation: IF-1, IF-2A, IF-3, EF-Tu or (Tu--Ts) and EF-G. The dependence of elongation on the Mg2+ concentration in the medium at RNA 4 translation has been determined.

Cell-Free System↗

PubMed1994

The cis-effect of a nascent peptide on its translating ribosome: influence of the cat-86 leader pentapeptide on translation termination at leader codon 6.

Inducible cat genes from Gram-positive bacteria are regulated by translation attenuation. The inducer chloramphenicol stalls a ribosome at a specific site in the leader of cat transcripts; this destabilizes a downstream stem-loop structure that normally sequesters the ribosome-binding site for the cat structural gene. The five-amino-acid peptide MVKTD that is synthesized when a ribosome has translated to the leader induction site is an inhibitor of peptidyl transferase in vitro. Thus, the peptide may be the in vivo determinant of the site of ribosome stalling. Here we provide evidence that the leader pentapeptide can exert a cis-effect on its translating ribosome in vivo. Converting leader codon 6 to the ochre codon results in expression of cat-86 in the absence of inducer. We term this autoinduction. Autoinduction is abolished by mutations that change the amino-acid sequence of the leader peptide but have no, or little, effect on the sequence of nucleotides at the leader stall site. In contrast, four nucleotide changes within the leader site occupied by the stalled ribosome that result in synonymous codon replacements do not diminish autoinduction. Our evidence indicates that the cat-86 leader pentapeptide can alter the function of its translating ribosome.

Amino Acid Sequence↗

PubMed1995

The sequence surrounding the translation initiation codon of the pea plastocyanin gene increases translational efficiency of a reporter gene.

The 5'-upstream region of the pea plastocyanin gene (petE) directed 5-10-fold higher levels of beta-glucuronidase (GUS) activity than the cauliflower mosaic virus 35S promoter in transgenic tobacco plants, although the levels of GUS mRNA were similar. The sequence (AAAAAUGG) around the translation initiation codon of petE enhanced translation of the GUS mRNA 10-fold compared to translation from the GUS translation initiation codon in transgenic tobacco plants and transfected protoplasts.

Base Sequence↗

PubMed1995

Almost the entire 5' non-translated region of hepatitis C virus is required for cap-independent translation.

To investigate which hairpin structures within the 5' untranslated region of hepatitis C virus (HCV) are necessary for cap-independent translation, mutants were constructed that lack one or more hairpin structures. Here we demonstrate, by constructing precisely defined hairpin deletion mutants, that with the exception of the most 5' located hairpin structure, which on deletion shows an increase on translation, each of the predicted hairpins is found to be essential for cap-independent translation. In addition, we demonstrate that HCV 5'UTR driven translation is stimulated by poliovirus 2Apro co-expression.

Animals↗

PubMed1985

Synthesis of surfactant-associated glycoprotein A by rat type II epithelial cells. Primary translation products and post-translational modification.

Surfactant-associated glycoproteins A, 38 (A3), 32 (A2) and 26 (A1) kDa, pI (4.2-4.8), were identified as related proteins present in surfactant isolated from rat lung lavage fluid. Differences in size and charge among surfactant-associated glycoproteins A were related to differences in glycosylation as determined by reduction of the larger forms (38 and 32 kDa) to 26 kDa by endoglycosidase F and by increased isoelectric points of the glycosylated forms after treatment with neuraminidase. Synthesis and secretion of surfactant-associated glycoproteins A and precursors were demonstrated in purified rat Type II epithelial cells by immunoprecipitation of [35S]methionine-labelled proteins with anti-surfactant-associated glycoprotein A antisera. In pulse-chase experiments, labelled proteins 26-34 kDa, appeared within 10 min and smaller forms co-migrated with surfactant-associated glycoprotein A from alveolar lavage. The relative abundance of the larger molecular mass forms (30-34 kDa, pI 4.8) increased at later times up to 3 h. More acidic mature forms, which co-migrated with surfactant-associated glycoproteins A2 and A3 in surfactant (38 and 32 kDa), were readily detectable in the media, but were not abundant forms in lysates of labelled Type II cells after 1-3 h of incubation. Primary translation products of surfactant-associated glycoprotein A were immunoprecipitated with monospecific anti-surfactant-associated glycoprotein A antiserum after in vitro translation of poly(A)+ mRNA isolated from adult rat lung. The immunoprecipitated translation product migrated at 26 kDa, pI 4.8, and migrated slightly faster than surfactant-associated glycoprotein A1 from surfactant. Treatment of surfactant-associated glycoprotein A with bacterial collagenase resulted in proteolytic fragments 23-20 kDa, pI 4.2-4.8, which no longer underwent sulfhydryl-dependent cross-linking, suggesting that the collagen-like domain was required for the sulfhydryl-dependent oligomerization. Surfactant-associated glycoproteins A are synthesized by rat Type II epithelial cells as pre-proteins, 26-34 kDa. Larger forms result primarily from N-linked glycosylation of the 26 kDa primary translation product. Mature, more acidic forms result from further addition of sialic acid.

Animals↗

PubMed1988

Translation mapping with the flavivirus Kunjin: gene order and anomalies in translation of Ns5.

Kunjin (KUN) virus-infected cells were synchronized in translation by reversal of hypertonic inhibition; cells were then pulse-labelled with [35S]methionine. Electrophoretic analyses defined the sequence of incorporation of label into all the known KUN gene products shown previously to be unique and unrelated. GP44 or NS1 was inadequately labelled for analysis and was assumed to be translated sequentially after E in the polyprotein sequence. The relationship of the KUN gene order obtained by translation mapping to that proposed by Rice et al. Science (1985) 229, 726-733 based on the nucleotide sequence of yellow fever virus is as follows: KUN: 5'-C.GP20.E.GP44.P19.P10.P71. (?).P21.P98-3' YF: 5'-C.prM.E.NS1.ns2a.ns2b.NS3.ns4a.ns4b. NS5-3'. These results eliminate the ambiguities in identities of the previously hypothetical ns2a, ns2b and ns4b. Although ns4a was not positively identified, a labelled protein of Mr 12,000 to 14,000 was observed in one experiment and it mapped in the appropriate position for ns4a. Variation occurred in translation of NS5 when the hypertonic treatment of 40 min at 37 degrees C was reduced in time or in temperature.

Animals↗

PubMed1986

Enhancement of translational efficiency by the Escherichia coli atpE translational initiation region: its fusion with two human genes.

The cDNA sequences encoding mature human interleukin 2 (IL2) and beta-interferon (INF beta), respectively, were fused with various translational initiation regions and inserted into two different types of expression vector. The relative levels of expression of the two genes and the functional stability of their respective mRNAs were examined in vivo in Escherichia coli hosts. The addition of the 30-bp sequence, found immediately upstream of the E. coli atpE gene Shine-Dalgarno (SD) sequence, to the translational initiation regions of IL2 and INF beta increased the expression of both these genes by a factor of 6-10. Thus this sequence, which naturally acts within the E. coli atp operon to enhance the translational initiation frequency of the atpE gene, can increase the expression of other genes in E. coli. It may exemplify a specific type of recognition signal for the E. coli translational apparatus.

Base Sequence↗

PubMed1998

The RNA recognition motif of yeast translation initiation factor Tif3/eIF4B is required but not sufficient for RNA strand-exchange and translational activity.

The Saccharomyces cerevisiae TIF3 gene encodes a 436-amino acid (aa) protein that is the yeast homologue of mammalian translation Initiation factor eIF4B. Tif3p can be divided into three parts, the N-terminal region with an RNA recognition motif (RRM) (aa 1-182), followed in the middle part by a sevenfold repeat of 26 amino acids rich in basic and acidic residues (as 183-350), and a C-terminal region without homology to any known sequence (aa 351-436). We have analyzed several Tif3 proteins with deletions at their N and C termini for their ability (1) to complement a tif3delta strain in vivo, (2) to stimulate Tif3-dependent translation extracts, (3) to bind to single-stranded RNA, and (4) to catalyze RNA strand-exchange in vitro. Here we report that yeast Tif3/eIF4B contains at least two RNA binding domains able to bind to single-stranded RNA. One is located in the N-terminal region of the protein carrying the RRM, the other in the C-terminal two-thirds region of Tif3p. The RRM-containing domain and three of the seven repeat motifs are essential for RNA strand-exchange activity of Tif3p and translation in vitro and for complementation of a tif3delta strain, suggesting an important role for RNA strand-exchange activity in translation.

Cell-Free System↗

PubMed1996

Strength of translation initiation signal sequence of mRNA as studied by quantification method: effect of nucleotide substitutions upon translation efficiency in rat preproinsulin mRNA.

Concerning the translation initiation signals in vertebrate mRNAs, both the ATG initiation codon and the sequences flanking the initiation codon are required to direct the position of initiation. A consensus sequence for the signal, (GCC)GCC(A or G)CCATGG, has been proposed, but actual initiation sequences differ from it to a greater or lesser degree. In the present report, the translation initiation signal sequences of rat preproinsulin and its mutant mRNAs were analyzed using a quantification method proposed previously. In this method, each 16 nt sequence in the mRNA was characterized by its sample score, which shows strength of the signal. So far, Kozak has constructed a number of preproinsulin mutant mRNAs in which nucleotides flanking the ATG codon are systematically varied, and measured the translation initiation efficiency in terms of the proinsulin product. Her experimental results were well understood on the basis of the strength of the translation initiation signal sequence.

Animals↗

PubMed1993

Translation elongation factor 3: a fungus-specific translation factor?

Fungi appear to be unique in their requirement for a third soluble translation elongation factor. This factor, designated elongation factor 3 (EF-3), was first described in the yeast Saccharomyces cerevisiae and has subsequently been identified in a wide range of fungal species including Candida albicans and Schizosaccharomyces pombe. EF-3 exhibits ribosome-dependent ATPase and GTPase activities that are not intrinsic to the fungal ribosome, but which are essential for translation elongation. Recent studies on the structure of EF-3 from several fungal species have shown that it consists of a repeated domain, with each domain containing the expected putative ATP- and GTP-binding motifs. Overall, EF-3 shows striking amino acid similarity to members of the ATP-binding Cassette (ABC) family of membrane-associated transport proteins although EF-3 is not itself directly membrane-associated. Regions of the EF-3 polypeptide also show structural homology with other translation-associated factors including aminoacyl-tRNA synthetases and the Escherichia coli ribosomal protein S5. While the precise role of EF-3 in the translation elongation cycle remains to be defined, recent evidence suggests that it may be involved in optimizing accuracy during mRNA decoding at the ribosomal A site. Furthermore, the essential nature of EF-3 with respect to the fungal cell indicates that it may be an effective antifungal target. Its apparently ubiquitous occurrence throughout the fungal kingdom also suggests that it may be a useful fungal taxonomic marker.

Amino Acid Sequence↗