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PubMed2005

Overexpression of the COX2 translational activator, Pet111p, prevents translation of COX1 mRNA and cytochrome c oxidase assembly in mitochondria of Saccharomyces cerevisiae.

Dramatically elevated levels of the COX2 mitochondrial mRNA-specific translational activator protein Pet111p interfere with respiratory growth and cytochrome c oxidase accumulation. The respiratory phenotype appears to be caused primarily by inhibition of the COX1 mitochondrial mRNA translation, a finding confirmed by lack of cox1Delta::ARG8(m) reporter mRNA translation. Interference with Cox1p synthesis depends to a limited extent upon increased translation of the COX2 mRNA, but is largely independent of it. Respiratory growth is partially restored by a chimeric COX1 mRNA bearing the untranslated regions of the COX2 mRNA, and by overproduction of the COX1 mRNA-specific activators, Pet309p and Mss51p. These results suggest that excess Pet111p interacts unproductively with factors required for normal COX1 mRNA translation. Certain missense mutations in PET111 alleviate the interference with COX1 mRNA translation but do not completely restore normal respiratory growth in strains overproducing Pet111p, suggesting that elevated Pet111p also perturbs assembly of newly synthesized subunits into active cytochrome c oxidase. Thus, this severe imbalance in translational activator levels appears to cause multiple problems in mitochondrial gene expression, reflecting the dual role of balanced translational activators in cooperatively regulating both the levels and locations of organellar translation.

Cyclooxygenase 1↗

PubMed2006

A new in vitro translation system for non-radioactive assay from tobacco chloroplasts: effect of pre-mRNA processing on translation in vitro.

We previously developed an in vitro translation system derived from tobacco chloroplasts. Here, we report a significantly improved in vitro translation system. By modifying preparation procedures for chloroplast extracts and reaction conditions, we achieved 100-fold higher translation activity than the previous system. The new system does not require the supplement of Escherichia coli tRNAs due to the omission of micrococcal nuclease treatment, thus the tRNA population reflects the intrinsic tRNA population in tobacco chloroplasts. The rate of translation initiation from a variety of chloroplast mRNAs may be measured by monitoring the fluorescence intensity of synthesized green fluorescent protein, which is a non-radioactive detection method. Incorporation of an amino acid linked to a fluorescent dye also allows detection of the translation products in vitro. Using our new system, we found that mRNAs carrying unprocessed or processed atpH and rbcL 5'-UTRs were efficiently translated at similar rates, whereas translation of mRNAs with processed atpB and psbB 5'-UTRs was more efficient than those with unprocessed 5'-UTRs. These results suggest that the role of 5'-UTR processing in the regulation of chloroplast gene expression differs between mRNAs. The new in vitro translation system will be a powerful tool to investigate the mechanism of chloroplast mRNA translation.

5' Untranslated Regions↗

PubMed1994

Vertebrate mRNAs with a 5'-terminal pyrimidine tract are candidates for translational repression in quiescent cells: characterization of the translational cis-regulatory element.

The translation of mammalian ribosomal protein (rp) mRNAs is selectively repressed in nongrowing cells. This response is mediated through a regulatory element residing in the 5' untranslated region of these mRNAs and includes a 5' terminal oligopyrimidine tract (5' TOP). To further characterize the translational cis-regulatory element, we monitored the translational behavior of various endogenous and heterologous mRNAs or hybrid transcripts derived from transfected chimeric genes. The translational efficiency of these mRNAs was assessed in cells that either were growing normally or were growth arrested under various physiological conditions. Our experiments have yielded the following results: (i) the translation of mammalian rp mRNAs is properly regulated in amphibian cells, and likewise, amphibian rp mRNA is regulated in mammalian cells, indicating that all of the elements required for translation control of rp mRNAs are conserved among vertebrate classes; (ii) selective translational control is not confined to rp mRNAs, as mRNAs encoding the naturally occurring ubiquitin-rp fusion protein and elongation factor 1 alpha, which contain a 5' TOP, also conform this mode of regulation; (iii) rat rpP2 mRNA contains only five pyrimidines in its 5' TOP, yet this mRNA is translationally controlled in the same fashion as other rp mRNAs with a 5' TOP of eight or more pyrimidines; (iv) full manifestation of this mode of regulation seems to require both the 5' TOP and sequences immediately downstream; and (v) an intact translational regulatory element from rpL32 mRNA fails to exert its regulatory properties even when preceded by a single A residue.

Animals↗

PubMed2005

Eukaryotic translational coupling in UAAUG stop-start codons for the bicistronic RNA translation of the non-long terminal repeat retrotransposon SART1.

Most eukaryotic cellular mRNAs are monocistronic; however, many retroviruses and long terminal repeat (LTR) retrotransposons encode multiple proteins on a single RNA transcript using ribosomal frameshifting. Non-long terminal repeat (non-LTR) retrotransposons are considered the ancestor of LTR retrotransposons and retroviruses, but their translational mechanism of bicistronic RNA remains unknown. We used a baculovirus expression system to produce a large amount of the bicistronic RNA of SART1, a non-LTR retrotransposon of the silkworm, and were able to detect the second open reading frame protein (ORF2) by Western blotting. The ORF2 protein was translated as an independent protein, not as an ORF1-ORF2 fusion protein. We revealed by mutagenesis that the UAAUG overlapping stop-start codon and the downstream RNA secondary structure are necessary for efficient ORF2 translation. Increasing the distance between the ORF1 stop codon and the ORF2 start codon decreased translation efficiency. These results are different from the eukaryotic translation reinitiation mechanism represented by the yeast GCN4 gene, in which the probability of reinitiation increases as the distance between the two ORFs increases. The translational mechanism of SART1 ORF2 is analogous to translational coupling observed in prokaryotes and viruses. Our results indicate that translational coupling is a general mechanism for bicistronic RNA translation.

Amino Acid Sequence↗

PubMed2005

Eukaryotic translation initiation factor 4E availability controls the switch between cap-dependent and internal ribosomal entry site-mediated translation.

Translation of m7G-capped cellular mRNAs is initiated by recruitment of ribosomes to the 5' end of mRNAs via eukaryotic translation initiation factor 4F (eIF4F), a heterotrimeric complex comprised of a cap-binding subunit (eIF4E) and an RNA helicase (eIF4A) bridged by a scaffolding molecule (eIF4G). Internal translation initiation bypasses the requirement for the cap and eIF4E and occurs on viral and cellular mRNAs containing internal ribosomal entry sites (IRESs). Here we demonstrate that eIF4E availability plays a critical role in the switch from cap-dependent to IRES-mediated translation in picornavirus-infected cells. When both capped and IRES-containing mRNAs are present (as in intact cells or in vitro translation extracts), a decrease in the amount of eIF4E associated with the eIF4F complex elicits a striking increase in IRES-mediated viral mRNA translation. This effect is not observed in translation extracts depleted of capped mRNAs, indicating that capped mRNAs compete with IRES-containing mRNAs for translation. These data explain numerous reported observations where viral mRNAs are preferentially translated during infection.

Animals↗

PubMed1996

Cis-acting elements and trans-acting factors for accurate translation of chloroplast psbA mRNAs: development of an in vitro translation system from tobacco chloroplasts.

Translational regulation is an important step of gene expression in chloroplasts. To analyze biochemical mechanisms of translational regulation unique to higher plant chloroplasts, an in vitro translation system has been developed from tobacco chloroplasts. Conditions for chloroplast extraction and the in vitro translation reaction have been optimized with a tobacco psbA-lacZ fusion mRNA. The in vitro system supports accurate translation of a variety of chloroplasts mRNAs. Using a series of mutant psbA mRNAs, we showed that three elements within the 5'-untranslated region of the mRNA are required for translation. Two of them are complementary to the 3'-terminus of chloroplast 16S rRNA (termed RBS1 and RBS2) and the other is an AU-rich sequence (UAAAUAAA) located between RBS1 and RBS2 and is termed the AU box. mRNA competition experiments using the in vitro translation reaction and gel mobility shift assays revealed the existence of a trans-acting factor(s) for translation and its possible interaction with the AU box. We propose a model for the initiation of psbA translation whereby RBS1 and RBS2 bind cooperatively to the 3'-end of 16S rRNA resulting in looping out of the AU box, which facilitates the interaction of a trans-acting factor(s).

Base Sequence↗

PubMed1987

AUU-to-AUG mutation in the initiator codon of the translation initiation factor IF3 abolishes translational autocontrol of its own gene (infC) in vivo.

We previously showed that Escherichia coli translation initiation factor IF3 regulates the expression of its own gene infC at the translational level in vivo. Here we create two alterations in the infC gene and test their effects on translational autocontrol of infC expression in vivo by measuring beta-galactosidase activity expressed from infC-lacZ gene fusions under conditions of up to 4-fold derepression or 3-fold repression of infC expression. Replacement of the infC promoter with the trp promoter deletes 120 nucleotides of the infC mRNA 5' to the translation initiation site without affecting autogenous translational control. Mutation of the unusual AUU initiator codon of infC to the more common AUG initiator codon abolishes translation initiation factor IF3-dependent repression and derepression of infC expression in vivo. These results establish the AUU initiator codon of infC as an essential cis-acting element in autogenous translational control of translation initiation factor IF3 expression in vivo.

Bacteriophage lambda↗

PubMed1997

A new translational regulator with homology to eukaryotic translation initiation factor 4G.

Translation initiation in eukaryotes is facilitated by the cap structure, m7GpppN (where N is any nucleotide). Eukaryotic translation initiation factor 4F (eIF4F) is a cap binding protein complex that consists of three subunits: eIF4A, eIF4E and eIF4G. eIF4G interacts directly with eIF4E and eIF4A. The binding site of eIF4E resides in the N-terminal third of eIF4G, while eIF4A and eIF3 binding sites are present in the C-terminal two-thirds. Here, we describe a new eukaryotic translational regulator (hereafter called p97) which exhibits 28% identity to the C-terminal two-thirds of eIF4G. p97 mRNA has no initiator AUG and translation starts exclusively at a GUG codon. The GUG-initiated open reading frame (907 amino acids) has no canonical eIF4E binding site. p97 binds to eIF4A and eIF3, but not to eIF4E. Transient transfection experiments show that p97 suppresses both cap-dependent and independent translation, while eIF4G supports both translation pathways. Furthermore, inducible expression of p97 reduces overall protein synthesis. These results suggest that p97 functions as a general repressor of translation by forming translationally inactive complexes that include eIF4A and eIF3, but exclude eIF4E.

Amino Acid Sequence↗

PubMed1994

Internal translation initiation on poliovirus RNA: further characterization of La function in poliovirus translation in vitro.

Initiation of poliovirus RNA translation by internal entry of ribosomes is believed to require the participation of trans-acting factors. The mechanism of action of these factors is poorly defined. The limiting amount of one of these factors, La protein, in rabbit reticulocyte lysates (RRL) has been postulated to partially explain the inefficient translation of poliovirus RNA in this system. To further characterize La activity in translation and to identify other potential limiting factors, we assayed the ability of La protein as well as purified initiation factors, eIF-2, guanine nucleotide exchange factor (GEF), eIF-4A, eIF-4B, eIF-4F, and eIF-3, to stimulate the synthesis of P1, the capsid precursor protein, in poliovirus type 1 (Mahoney) RNA-programmed RRL. Of the proteins tested, only La, GEF, and to some extent eIF-2 stimulated the synthesis of P1. The enhanced translation of P1 in response to La occurred concomitantly with the inhibition of synthesis of most aberrant polypeptides, resulting from initiation in the middle of the genome. Deletion of the carboxy-terminal half (214 amino acids) of La did not decrease its binding to the poliovirus 5' untranslated region but abrogated the stimulatory and correcting activity in translation. In contrast to La, GEF and eIF-2 stimulated the overall translation and increased the synthesis of aberrant products as well as P1. Neither La, GEF, nor any other factor stimulated translation of encephalomyocarditis virus RNA in RRL. The implications of these findings for the mechanism of internal translation initiation on picornavirus RNAs are discussed.

Animals↗

PubMedDate not supplied

Inability of Agrobacterium tumefaciens ribosomes to translate in vivo mRNAs containing non-Shine-Dalgarno translational initiators.

Numerous data accumulated during the last decade have shown that the Shine-Dalgarno (SD) sequence is not a unique initiator of translation for Escherichia coli. Several other sequences, mostly of viral origin, have demonstrated their capability of either enhancing or initiating translation in vivo. A phage T7 gene 10 sequence, called "epsilon" (epsilon), has shown its high enhancing activity on translation in both Escherichia coli and Agrobacterium tumefaciens cells. In this study the epsilon, together with three other nucleotide sequences derived from the 5' non-translated regions of tobacco mosaic virus (TMV), papaya mosaic virus (PMV) and clover yellow mosaic virus (CYMV) RNAs are tested for translation initiation activity in A. tumefaciens cells. The obtained results indicate that none of them was capable of initiating translation in vivo of chloramphenicol acetyltransferase (CAT) mRNA. To determine whether their inactivity was related with structural differences in the ribosomal protein S1, the rpsA gene (coding for S1 protein in E. coli) was co-expressed in A. tumefaciens together with the cat gene placed under the translational control of the above sequences. Our results showed that the rpsA gene product did not make any of the four viral enhancers active in A. tumefaciens cells. The inability of A. tumefaciens ribosomes to translate mRNAs devoid of SD sequences indicates for a substantial difference in the ribosome structure of the two Gram negative bacteria E. coli and A. tumefaciens.

5' Untranslated Regions↗

PubMed1998

Role of the 3'-untranslated region of RPE65 mRNA in the translational regulation of the RPE65 gene: identification of a specific translation inhibitory element.

Previously, we demonstrated that explanted bovine retinal pigment epithelium (RPE) cells lose RPE65 protein, a major microsomal protein specific to RPE, while the RPE65 mRNA remains, suggesting posttranscriptional regulation of RPE65 expression in vitro. Accordingly, we analyze here the effect of the 5'- and 3'-untranslated regions (UTRs) of RPE65 mRNA on translational efficiency using in vitro translation systems. We compared the levels of translation products and mRNA stability among RPE65 transcripts containing deletions of the 5'- and 3'-UTRs. First, the 5'-UTR does not affect translational efficiency. However, the 3'-UTR does influence translation efficiency. A putative translation inhibitory element (TIE) is contained within the 170-nucleotide (nt) sequence downstream of the stop codon. There is also a weak destabilizing effect that is associated with the region 3' to the putative TIE. But the effect of this is much less than that of the TIE. This TIE, however, does not inhibit translation of the heterologous chloramphenicol acetyltransferase gene, suggesting that a specific interaction with the upstream RPE65 coding sequence, or its product, may be required. Thus, the posttranscriptional regulation of RPE65 mRNA expression observed in cultured RPE may be via a mechanism of translational inhibition.

Animals↗

PubMed2002

Autogenous translational inhibition of core protein: implication for switch from translation to RNA replication in hepatitis C virus.

Positive-stranded viruses use the genomic RNA as a common template for translation and RNA replication which proceed in inverse direction; a certain regulatory mechanism for translation control is probably required to coordinate these two antagonistic processes. Hepatitis C virus (HCV) core protein is a good candidate that might play a role in such a regulation. In this study, we further investigated whether HCV core protein modulates internal ribosome entry site (IRES)-directed translation. The inclusion of the core-coding sequence significantly suppressed translation initiated by HCV IRES in monocistronic and bicistronic reporter systems. The region mainly responsible for this inhibition was mapped to nt 441-473 of the core-coding sequence. This suppression was eliminated by frameshift mutations introduced into this region, suggesting that it is the core protein expressed in cis, rather than the core-coding nucleotide sequence that negatively modulates the efficiency of HCV IRES-dependent translation. Furthermore, the core protein provided in trans also specifically decreased the IRES activity in directing cap-independent translation both in transfected cells and in cell-free translation study. Consistently, a gel mobility shift assay showed a specific interaction between the core protein and HCV IRES-containing RNA transcript. These findings suggest that HCV core protein may down-regulate the cap-independent translation as a regulatory mechanism required for initiation of transcription.

5' Untranslated Regions↗

PubMed1992

Translational head movements of pigeons in response to a rotating pattern: characteristics and tool to analyse mechanisms underlying detection of rotational and translational optical flow.

Pigeons freely standing in the centre of a two-dimensionally textured cylinder not only rotate but also laterally translate their head in response to the pattern sinusoidally oscillating or unidirectionally rotating around their vertical axis. The translational head movement dominates the response at high oscillation frequencies, whereas in a unidirectionally rotating drum head translation declines at about the same rate as the rotational response increases. It is suggested that this is a consequence of charging the 'velocity storage' in the vestibulo-ocular system. Similar to the rotational head movement (opto-collic reflex), the translational head movement is elicited via a wide-field motion sensitive system. The underlying mechanism can be described as vector integration of movement vectors tangential to the pattern rotation. Stimulation of the frontal visual field elicits largest translational responses while rotational responses can be elicited equally well from any azimuthal position of a moving pattern. Experiments where most of the pattern is occluded by a screen and the pigeon is allowed to view the stimulus through one or two windows demonstrate a short-range inhibition and long-range excitation between movement detectors that feed into the rotational system. Furthermore, the results obtained from such types of experiments suggest that the rotational system inhibits the translational system. These mechanisms may help the pigeon to decompose image flow into its translational and rotational components. Because of their translational response to a rotational stimulus, it is concluded, however, that pigeons either generally cannot perfectly perform the task or they need further visual information, like differential image motion, that was not available to them in the paradigms.

Animals↗

PubMed1992

Messenger RNA secondary structure and translational coupling in the Escherichia coli operon encoding translation initiation factor IF3 and the ribosomal proteins, L35 and L20.

The Escherichia coli infC-rpmI-rplT operon encodes translation initiation factor IF3 and the ribosomal proteins, L35 and L20, respectively. The expression of the last cistron (rplT) has been shown to be negatively regulated at a post-transcriptional level by its own product, L20, which acts at an internal operator located within infC. The present work shows that L20 directly represses the expression of rpmI, and indirectly that of rplT, via translational coupling with rpmI. Deletions and an inversion of the coding region of rpmI, suggest an mRNA secondary structure forming between sequences within rpmI and the translation initiation site of rplT. To verify the existence of this structure, detailed analyses were performed using chemical and enzymatic probes. Also, mutants that uncoupled rplT expression from that of rpmI, were isolated. The mutations fall at positions that would base-pair in the secondary structure. Our model is that L20 binds to its operator within infC and represses the translation of rpmI. When the rpmI mRNA is not translated, it can base-pair with the ribosomal binding site of rplT, sequestering it, and abolishing rplT expression. If the rpmI mRNA is translated, i.e. covered by ribosomes, the inhibitory structure cannot form leaving the translation initiation site of rplT free for ribosomal binding and for full expression. Although translational coupling in ribosomal protein operons has been suspected to be due to the formation of secondary structures that sequester internal ribosomal binding sites, this is the first time that such a structure has been shown to exist.

Bacterial Proteins↗

PubMed2005

An efficient mammalian cell-free translation system supplemented with translation factors.

Development of an efficient cell-free translation system from mammalian cells is an important goal. We examined whether supplementation of HeLa cell extracts with any translation initiation factor or translational regulator could enhance protein synthesis. eIF2 (eukaryotic translation initiation factor 2) and eIF2B augmented translation of capped, uncapped and encephalomyocarditis virus-internal ribosome entry site-promoted mRNAs. eIF4E specifically stimulated capped mRNA translation, while p97, a homologue to the C-terminal two-thirds of eIF4G, increased uncapped mRNA translation. When the HeLa cell extract was supplemented with a combination of eIF2, eIF2B, and p97, the capacity to synthesize a protein from an uncapped mRNA became comparable to that from the capped counterpart stimulated with a combination of eIF2, eIF2B, and eIF4E. A dialysis method rendered the HeLa cell extract capable of synthesizing proteins for 36h, and the yield was augmented when supplemented with initiation factors. In contrast, the productivity of a rabbit reticulocyte lysate was not enhanced by this method. Collectively, the translation factor-supplemented HeLa cell extract should become an important tool for the production of recombinant proteins.

Cell-Free System↗

PubMed1998

Poly(A)-tail-promoted translation in yeast: implications for translational control.

The cap structure and the poly(A) tail synergistically activate mRNA translation in vivo. Recent work using Saccharomyces cerevisiae spheroplasts and a yeast cell-free translation system revealed that the poly(A) tail can function as an independent promotor for ribosome recruitment, to internal initiation sites within an mRNA. This raises the question of how regulatory upstream open reading frames and translational repressor proteins binding to the 5'UTR can function, as well as how regulated polyadenylation can support faithful activation of protein synthesis. We investigated the function of the regulatory upstream open reading frame 4 from the yeast GCN 4 gene and the effect of IRP-1 binding to an iron-responsive element introduced into the 5' UTR of reporter mRNAs. Both manipulations effectively block cap-dependent translation, whereas ribosome recruitment promoted by the poly(A) tail under non-competitive conditions can efficiently bypass both blocks. We show that the synergistic use of both, the cap structure and the poly-A tail enforced by mRNA competition reinstates the full extent of translational control by both types of 5' UTR regulatory elements. With a view towards regulated polyadenylation, we studied the function of poly(A) tails of defined length on the translation of capped mRNAs. We find that poly(A) tail elongation increases translational efficiency, particularly under competitive conditions. Our results integrate recent findings on the function of the poly(A) tail into an understanding of translational control.

5' Untranslated Regions↗

PubMed1995

The relationship between eukaryotic translation and mRNA stability. A short upstream open reading frame strongly inhibits translational initiation and greatly accelerates mRNA degradation in the yeast Saccharomyces cerevisiae.

A new strategy was developed to study the relationship between the translation and degradation of a specific mRNA in the yeast Saccharomyces cerevisiae. A series of 5'-untranslated regions (UTR) was combined with the cat gene from the bacterial transposon Tn9, allowing us to test the influence of upstream open reading frames (uORFs) on translation and mRNA stability. The 5'-UTR sequences were designed so that the minimum possible sequence alteration, a single nucleotide substitution, could be used to create a 7-codon ORF upstream of the cat gene. The uORF was translated efficiently, but at the same time inhibited translation of the cat ORF and destabilized the cat mRNA. Investigations of various derivatives of the 5'-UTR indicated that cat translation was primarily attributable to leaky scanning of ribosomes past the uORF rather than to reinitiation. Therefore, these data directly demonstrate destabilization of a specific mRNA linked to changes in translational initiation on the same transcript. In contrast to the previously proposed nonsense-mediated mRNA decay pathway, destabilization was not triggered by premature translational termination in the main ORF and was not discernibly dependent upon a reinitiation-driven mechanism. This suggests the existence of an as yet not described pathway of translation-linked mRNA degradation.

Base Sequence↗

PubMed1999

Opposite translational control of GLUT1 and GLUT4 glucose transporter mRNAs in response to insulin. Role of mammalian target of rapamycin, protein kinase b, and phosphatidylinositol 3-kinase in GLUT1 mRNA translation.

Prolonged exposure of 3T3-L1 adipocytes to insulin increases GLUT1 protein content while diminishing GLUT4. These changes arise in part from changes in mRNA transcription. Here we examined whether there are also specific effects of insulin on GLUT1 and GLUT4 mRNA translation. Insulin enhanced association of GLUT1 mRNA with polyribosomes and decreased association with monosomes, suggesting increased translation. Conversely, insulin arrested the majority of GLUT4 transcripts in monosomes. Insulin inactivates the translational suppressor eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) through the mammalian target of rapamycin (mTOR). Hence, we examined the effect of rapamycin on GLUT1 mRNA translation and protein expression. Rapamycin abrogated the insulin-mediated increase in GLUT1 protein synthesis through partial inhibition of GLUT1 mRNA translation and partial inhibition of the rise in GLUT1 mRNA. 4E-BP1 inhibited GLUT1 mRNA translation in vitro. Because phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB), in concert with mTOR, inactivate 4E-BP1, we explored their role in GLUT1 protein expression. Cotransfection of cytomegalovirus promoter-driven, hemagglutinin epitope-tagged GLUT1 with dominant inhibitory mutants of PI3K or PKB inhibited the insulin-elicited increase in hemagglutinin-tagged GLUT1 protein. These results unravel the opposite effects of insulin on GLUT1 and GLUT4 mRNA translation. Increased GLUT1 mRNA translation appears to occur via the PI3K/PKB/mTOR/4E-BP1 cascade.

3T3 Cells↗