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At least 19 recordsLinked to original sources

PubMed1990

Frog virus 3-mediated translational shut-off: frog virus 3 messages are translationally more efficient than host and heterologous viral messages under conditions of increased translational stress.

Frog virus 3 rapidly and selectively blocks host cell translation while synthesizing more than 60 virus-specific polypeptides. Previous work indicated that virus infection led to activation of a kinase that phosphorylated and, as a consequence, inactivated eIF-2. Although phosphorylation of eIF-2 could explain the rapid decline in host cell translation, it could not explain how viral protein synthesis persisted in the face of host shut-off. To explain this phenomenon, we speculated that viral messages, either as a consequence of their higher translational efficiency or their greater abundance, were able to outcompete host messages for the remaining translational initiation complexes. To test this hypothesis, the relative translational efficiency of three characteristic FV3 messages was measured against that of several model messages. Translational efficiency was determined by monitoring the resistance (and hence the competitiveness) of a given transcript to increasing concentrations of salt in vitro and in vivo. In both rabbit reticulocyte lysates and wheat germ extracts, FV3 messages were more resistant to supra-optimal concentrations of potassium acetate than globin message and three BMV transcripts. In vivo, FV3 polypeptides were synthesized in the presence of salt concentrations that blocked host cell protein synthesis. These results suggest that the selective translation of FV3 messages in virus-infected cells may partly be due to the higher translational efficiency of viral messages. Structural features that contribute to translational efficiency are discussed.

Acetates↗

PubMed2006

Translation control of trpG from transcripts originating from the folate operon promoter of Bacillus subtilis is influenced by translation-mediated displacement of bound TRAP, while translation control of transcripts originating from a newly identified trpG promoter is not.

Bacillus subtilis trpG encodes a glutamine amidotransferase subunit that participates in the biosynthesis of both tryptophan and folic acid. TRAP inhibits translation of trpG in response to tryptophan by binding to a site that overlaps the trpG Shine-Dalgarno sequence, thereby blocking ribosome binding. Similar mechanisms regulate trpP and ycbK translation. The equilibrium binding constants of tryptophan-activated TRAP for the trpG, ycbK, and trpP transcripts were determined to be 8, 3, and 50 nM, respectively. Despite TRAP having a higher affinity for the trpG transcript, TRAP exhibited the least control of trpG expression. The trpG Shine-Dalgarno sequence overlaps the stop codon of the upstream pabB gene, while six of nine triplet repeats within the TRAP binding site are located upstream of the pabB stop codon. Thus, ribosomes translating the upstream pabB cistron could be capable of reducing TRAP-dependent control of TrpG synthesis by displacing bound TRAP. Expression studies using pabB-trpG'-'lacZ fusions in the presence or absence of an engineered stop codon within pabB suggest that translation-mediated displacement of bound TRAP reduces TRAP-dependent inhibition of TrpG synthesis from transcripts originating from the folate operon promoter (P(pabB)). A new trpG promoter (P(trpG)) was identified in the pabB coding sequence that makes a larger contribution to trpG expression than does P(pabB). We found that TRAP-dependent regulation of trpG expression is more extensive for a transcript originating from P(trpG) and that transcripts originating from P(trpG) are not subject to translation-mediated displacement of bound TRAP.

Anthranilate Synthase↗

PubMed1995

The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4 gamma and the translational repressors 4E-binding proteins.

Eukaryotic translation initiation factor 4E (eIF-4E), which possesses cap-binding activity, functions in the recruitment of mRNA to polysomes as part of a three-subunit complex, eIF-4F (cap-binding complex). eIF-4E is the least abundant of all translation initiation factors and a target of growth regulatory pathways. Recently, two human cDNAs encoding novel eIF-4E-binding proteins (4E-BPs) which function as repressors of cap-dependent translation have been cloned. Their interaction with eIF-4E is negatively regulated by phosphorylation in response to cell treatment with insulin or growth factors. The present study aimed to characterize the molecular interactions between eIF-4E and the other subunits of eIF-4F and to similarly characterize the molecular interactions between eIF-4E and the 4E-BPs. A 49-amino-acid region of eIF-4 gamma, located in the N-terminal side of the site of cleavage by Picornaviridae protease 2A, was found to be sufficient for interacting with eIF-4E. Analysis of deletion mutants in this region led to the identification of a 12-amino-acid sequence conserved between mammals and Saccharomyces cerevisiae that is critical for the interaction with eIF-4E. A similar motif is found in the amino acid sequence of the 4E-BPs, and point mutations in this motif abolish the interaction with eIF-4E. These results shed light on the mechanisms of eIF-4F assembly and on the translational regulation by insulin and growth factors.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Translation initiation with GUC codon in the archaeon Halobacterium salinarum: implications for translation of leaderless mRNA and strict correlation between translation initiation and presence of mRNA.

We have investigated whether anticodon sequence mutant of an archaeal initiator tRNA can initiate protein synthesis using reporter genes carrying mutations in the initiation codon. Halobacterium salinarum was used as the model organism and the bacterio-opsin gene (bop), which encodes the precursor of the protein component of the purple membrane protein bacterio-opsin (Bop), was chosen as the reporter. We demonstrate that a CAU to GAC anticodon sequence mutant of Haloferax volcanii initiator tRNA can initiate Bop protein synthesis using GUC as the initiation codon in H. salinarum. We generated four mutant bop genes, each carrying the AUG to GUC initiation codon mutation, with or without a compensatory mutation to maintain a predicted stem-loop structure at the 5'-end of the bop mRNA, and with or without mutations to test translation initiation at a site corresponding to the amino terminus of mature bacterio-opsin. H. salinarum chromosomal recombinants containing these mutant genes were phenotypically Pum- (purple membrane negative). Upon transformation with a plasmid carrying the mutant initiator tRNA gene, only strains designed to maintain the bop mRNA stem-loop structure produced Bop and were phenotypically Pum+ as indicated by purple colony colour, and immunoblotting and spectral analysis of cell extracts. Thus GUC can serve as an initiation codon in archaea and the stem-loop structure in the bop mRNA is important for translation. Interestingly, for the same mutant mRNA, only transformants that produce Bop protein contain bop mRNA. These results suggest either a strong coupling between translation and mRNA stability or strong transcriptional polarity in H. salinarum.

5' Untranslated Regions↗

PubMed1990

Pre-translational, translational, and post-translational mechanisms in adaptation of intestinal proteins.

The enterocyte exhibits remarkable short-term adaptation to a meal, complementing the well recognized adaptation of the stomach and pancreas. The mechanisms utilized include increased protein synthesis by pre-translational and translational events, and post-translational alterations in protein degradation and secretion. All of these events occur within a few hours after the meal. These adaptations are compared with the more long-term events in post-natal development that occur over a period of days, at least in the rat. They contrast even more with the macroscopic changes that also occur over days and weeks following intestinal resection, the model most commonly associated with the concept of intestinal adaptation. The demonstration of cellular and molecular adaptation within a few hours of a meal represents an expansion of the definition of intestinal adaptation and may provide clues to the explanation of more delayed events.

Adaptation, Physiological↗

PubMed2000

Eukaryote-specific domains in translation initiation factors: implications for translation regulation and evolution of the translation system.

Computational analysis of sequences of proteins involved in translation initiation in eukaryotes reveals a number of specific domains that are not represented in bacteria or archaea. Most of these eukaryote-specific domains are known or predicted to possess an alpha-helical structure, which suggests that such domains are easier to invent in the course of evolution than are domains of other structural classes. A previously undetected, conserved region predicted to form an alpha-helical domain is delineated in the initiation factor eIF4G, in Nonsense-mediated mRNA decay 2 protein (NMD2/UPF2), in the nuclear cap-binding CBP80, and in other, poorly characterized proteins, which is named the NIC (NMD2, eIF4G, CBP80) domain. Biochemical and mutagenesis data on NIC-containing proteins indicate that this predicted domain is one of the central adapters in the regulation of mRNA processing, translation, and degradation. It is demonstrated that, in the course of eukaryotic evolution, initiation factor eIF4G, of which NIC is the core, conserved portion, has accreted several additional, distinct predicted domains such as MI (MA-3 and eIF4G ) and W2, which probably was accompanied by acquisition of new regulatory interactions.

Adaptor Proteins, Signal Transducing↗

PubMed1991

Sequences mediating the translation of mouse S16 ribosomal protein mRNA during myoblast differentiation and in vitro and possible control points for the in vitro translation.

The translation of ribosomal protein (r-protein) mRNAs is generally inefficient and regulated during the differentiation of mouse myoblasts into fibers. In this discussion we show that the first 31 nucleotides of the S16 r-protein mRNA, when located at the 5' end of the mRNA, are sufficient to impart the translational properties of an r-protein mRNA to the SV-GALK mRNA, which is normally translated efficiently in both myoblasts and fibers. If the same S16 sequences are located within the interior of the 5'-untranslated region of the SV-GALK mRNA, however, they do not impart the translational properties of an r-protein mRNA to the SV-GALK mRNA. The translation of mouse r-protein mRNAs was examined in vitro to help elucidate the mechanisms controlling their translation. Mouse r-protein mRNAs are inefficiently translated in rabbit reticulocyte extracts, and the same sequences that mediate their inefficient and regulated translation during myoblast differentiation also mediate their inefficient translation in a position-dependent manner in reticulocyte extracts. To determine whether the subpolysomal r-protein mRNAs that are not actively translated in vivo are capable of translation, subpolysomal RNA was translated in reticulocyte extracts. The subpolysomal r-protein mRNAs are just as capable of translation as are polysomal mRNAs. To help identify the initiation factors and/or the steps in the initiation pathway that mediate the inefficient translation of r-protein mRNAs, reticulocyte extracts were supplemented with purified initiation factors. Only eIF-4F, the cap-binding complex, and eIF-3, which is involved in subunit dissociation and interacts with eIF-4F during initiation, stimulated the translation of r-protein mRNA. These experiments, along with m7GDP inhibition studies, suggest that eIF-4F and/or eIF-3, or the steps mediated by these factors, mediate the inefficient translation in reticulocyte extracts and raise the possibility that these steps also control the regulated translation of r-protein mRNAs during myoblast differentiation.

Animals↗

PubMed1991

Mechanism of selective translation of vaccinia virus mRNAs: differential role of poly(A) and initiation factors in the translation of viral and cellular mRNAs.

We have recently demonstrated that the poly(A) moieties of short RNAs obtained from both in vitro transcription and from vaccinia virus (VV)-infected cells exhibit dissimilar effects on the in vitro translation of cellular and VV mRNAs (R. Bablanian, G. Coppola, P. Masters, and A. K. Banerjee, Virology 148:375-380, 1986; M. J. Su and R. Bablanian, Virology 179:679-693, 1990). In the present study, we have investigated the roles of poly(A), m7GTP, and initiation factors in the mechanism of selective translation of VV mRNAs. The effects of unfractionated poly(A) [termed poly(A)un, with various chain lengths up to 3,000 nucleotides] and a 150- to 300-nucleotide fraction of synthetic poly(A) [termed poly(A)150-300] on the translation of HeLa cell mRNAs and early and late VV mRNAs were studied. Both the poly(A)un and the poly(A)150-300 completely inhibited the translation of HeLa cell mRNAs obtained from total cytoplasmic RNA in the nuclease-treated reticulocyte lysates. Viral mRNAs from total cytoplasmic RNA also were slightly inhibited (15 to 38%) by the poly(A)un, whereas the poly(A)150-300 had no significant effect on their translation. The translation of oligo(dT)-cellulose-selected HeLa mRNAs was as sensitive to inhibition by poly(A)150-300 as the mRNAs found in total cytoplasmic RNA. However, the translations of oligo(dT)-cellulose-selected viral mRNAs become more sensitive to the inhibitory effect of poly(A)150-300 than the translations of viral mRNAs found in the total cytoplasmic RNA. Both HeLa and VV mRNAs became more resistant to the poly(A)-mediated inhibition when these mRNAs were deadenylated, but the relative resistance to inhibition by poly(A)150-300 of deadenylated VV mRNAs was much greater than that of HeLa cell mRNAs. The translation of VV mRNAs was significantly less inhibited than the translation of HeLa mRNAs when the cap analog, m7GTP, was added to the cell-free system. The inhibition of HeLa cell mRNA translation by both poly(A)un and poly(A)150-300 was completely restored when poly(A)-binding protein (PAB) was added to the cell-free translational system. The addition of eukaryotic initiation factor 4A (eIF-4A) did not restore translation when poly(A)un was used to inhibit translation; however, inhibition by poly(A)150-300 was significantly reversed by this initiation factor. The reversal of poly (A)-mediated inhibition of HeLa cell mRNA translation was additive when PAB was used together with eIF-4A. Early VV mRNA translation was only slightly inhibited by poly(A)un (15%), and this inhibition was completely reversed by either PAB or eIF-4A.(ABSTRACT TRUNCATED AT 400 WORDS)

Carrier Proteins↗

PubMed2005

Eukaryotic translation initiation factor 4GI and p97 promote cellular internal ribosome entry sequence-driven translation.

Numerous cellular mRNAs encoding proteins critical during cell stress, apoptosis, and the cell cycle seem to be translated by means of internal ribosome entry sequences (IRES) when cap-dependent translation is compromised. The underlying molecular mechanisms are largely unknown. Using a HeLa-based cell-free translation system that mirrors the function of cellular IRESs in vitro, we recently demonstrated that translation from the c-myc IRES continues after proteolytic cleavage of eukaryotic translation initiation factor (eIF) 4G. To address the role of eIF4G in cellular IRES-driven translation directly, we immunodepleted eIF4GI from the HeLa cell translation extracts. After efficient depletion of eIF4GI (>90%), both cap-dependent and c-myc IRES-dependent translations are diminished to residual levels (<5%). In striking contrast to cap-dependent translation, c-myc IRES-dependent translation is fully restored by addition of the conserved middle fragment of eIF4GI, harboring the eIF3- and eIF4A-binding sites. p97, an eIF4G-related protein that has been described both as an inhibitor of translation and as a modulator of apoptosis, not only suffices to also rescue c-myc IRES-driven (but not cap-dependent) translation, but it even superinduces IRES-mediated translation 3-fold compared with nondepleted extracts. Interestingly, both p97 and the middle fragment of eIF4GI also rescue translation driven by proapoptotic (p97) and antiapoptotic [X-linked inhibitor of apoptosis (XIAP) and cellular inhibitor of apoptosis 1 (c-IAP1)] IRESs, reflecting a broader role of these polypeptides in cellular IRES-mediated translation and indicating their importance in apoptosis.

5' Untranslated Regions↗

PubMed2000

A mutant allele of essential, general translation initiation factor DED1 selectively inhibits translation of a viral mRNA.

Positive-strand RNA virus genomes are substrates for translation, RNA replication, and encapsidation. To identify host factors involved in these functions, we used the ability of brome mosaic virus (BMV) RNA to replicate in yeast. We report herein identification of a mutation in the essential yeast gene DED1 that inhibited BMV RNA replication but not yeast growth. DED1 encodes a DEAD (Asp-Glu-Ala-Asp)-box RNA helicase required for translation initiation of all yeast mRNAs. Inhibition of BMV RNA replication by the mutant DED1 allele (ded1-18) resulted from inhibited expression of viral polymerase-like protein 2a, encoded by BMV RNA2. Inhibition of RNA2 translation was selective, with no effect on general cellular translation or translation of BMV RNA1-encoded replication factor 1a, and was independent of p20, a cellular antagonist of DED1 function in translation. Inhibition of RNA2 translation in ded1-18 yeast required the RNA2 5' noncoding region (NCR), which also conferred a ded1-18-specific reduction in expression on a reporter gene mRNA. Comparison of the similar RNA1 and RNA2 5' NCRs identified a 31-nucleotide RNA2-specific region that was required for the ded1-18-specific RNA2 translation block and attenuated RNA2 translation in wild-type yeast. Further comparisons and RNA structure predictions suggest a modular arrangement of replication and translation signals in RNA1 and RNA2 5' NCRs that appears conserved among bromoviruses. The 5' attenuator and DED1 dependence of RNA2 suggest that, despite its divided genome, BMV regulates polymerase translation relative to other replication factors, just as many single-component RNA viruses use translational read-through and frameshift mechanisms to down-regulate polymerase. The results show that a DEAD-box helicase can selectively activate translation of a specific mRNA and may provide a paradigm for translational regulation by other members of the ubiquitous DEAD-box RNA helicase family.

Base Sequence↗

PubMed2003

Down-regulation of the mitochondrial translation system during terminal differentiation of HL-60 cells by 12-O-tetradecanoyl-1-phorbol-13-acetate: comparison with the cytoplasmic translation system.

Mitochondrial (mt) biogenesis depends on both the nuclear and mt genomes, and a coordination of these two genetic systems is necessary for proper cell functioning. Little is known about the regulatory mechanisms of mt translation or about the expression of mt translation factors. Here, we studied the expression of mt translation factors during 12-O-tetradecanoyl-1-phorbol-13-acetate (TPA)-induced terminal differentiation of HL-60 cells. For all mt translation factors investigated, mRNA expression was markedly down-regulated in a coordinate and specific manner, whereas mRNA levels for the cytoplasmic translation factors showed only a slight reduction. An actinomycin D chase study and nuclear run-on assay revealed that the TPA-induced decrease in mt elongation factor Tu (EF-Tumt) mRNA mainly results from decreased mRNA stability. Polysome analysis showed that there was no significant translational control of mt translation factor (EF-Tumt, ribosomal proteins L7/L12mt and S12mt) mRNA expression during differentiation. Thus, the decreased protein level of one of these mt translation factors (EF-Tumt) simply reflects its decreased mRNA level. It was also demonstrated by pulse labeling of mt translation products that the down-regulation of mt translational activity is actually associated with down-regulated mt translation factor expression during cellular differentiation. Our results illustrate that the regulatory mechanisms of mt translational activity upon terminal differentiation (in response to the growth arrest) is different to that of the cytoplasmic system, where the control of mRNA translational efficiency of major translation factors is the central mechanism for their down-regulation.

Blotting, Northern↗

PubMed2001

Translational silencing of ceruloplasmin requires the essential elements of mRNA circularization: poly(A) tail, poly(A)-binding protein, and eukaryotic translation initiation factor 4G.

Ceruloplasmin (Cp) is a glycoprotein secreted by the liver and monocytic cells and probably plays roles in inflammation and iron metabolism. We showed previously that gamma interferon (IFN-gamma) induced Cp synthesis by human U937 monocytic cells but that the synthesis was subsequently halted by a transcript-specific translational silencing mechanism involving the binding of a cytosolic factor(s) to the Cp mRNA 3' untranslated region (UTR). To investigate how protein interactions at the Cp 3'-UTR inhibit translation initiation at the distant 5' end, we considered the "closed-loop" model of mRNA translation. In this model, the transcript termini are brought together by interactions of poly(A)-binding protein (PABP) with both the poly(A) tail and initiation factor eIF4G. The effect of these elements on Cp translational control was tested using chimeric reporter transcripts in rabbit reticulocyte lysates. The requirement for poly(A) was shown since the cytosolic inhibitor from IFN-gamma-treated cells minimally inhibited the translation of a luciferase reporter upstream of the Cp 3'-UTR but almost completely blocked the translation of a transcript containing a poly(A) tail. Likewise, a requirement for poly(A) was shown for silencing of endogenous Cp mRNA. We considered the possibility that the cytosolic inhibitor blocked the interaction of PABP with the poly(A) tail or with eIF4G. We found that neither of these interactions were inhibited, as shown by immunoprecipitation of PABP followed by quantitation of the poly(A) tail by reverse transcription-PCR and of eIF4G by immunoblot analysis. We considered the alternate possibility that these interactions were required for translational silencing. When PABP was depleted from the reticulocyte lysate with anti-human PABP antibody, the cytosolic factor did not inhibit translation of the chimeric reporter, thus showing the requirement for PABP. Similarly, in lysates treated with anti-human eIF4G antibody, the cytosolic extract did not inhibit the translation of the chimeric reporter, thereby showing a requirement for eIF4G. These data show that translational silencing of Cp requires interactions of three essential elements of mRNA circularization, poly(A), PABP, and eIF4G. We suggest that Cp mRNA circularization brings the cytosolic Cp 3'-UTR-binding factor into the proximity of the translation initiation site, where it silences translation by an undetermined mechanism. These results suggest that in addition to its important function in increasing the efficiency of translation, transcript circularization may serve as an essential structural determinant for transcript-specific translational control.

3' Untranslated Regions↗

PubMed2000

Kinetic proofreading scanning models for eukaryotic translational initiation: the cap and poly(A) tail dependency of translation.

Two simplified kinetic proofreading scanning (KPS) models were proposed to describe the 5' cap and 3' poly(A) tail dependency of eukaryotic translation initiation. In Model I, the initiation factor complex starts scanning and unwinding the secondary structure of the 5' untranslated region (UTR) from the 5' terminus of mRNA. In Model II, the initiation factor complex starts scanning from any binding site in the 5' UTR. In both models, following ATP hydrolysis, the initiation factor complex either dissociates from mRNA or continues to scan and unwind RNA secondary structure in the 5' UTR. This step repeats n times until the AUG codon is reached. These two models show very different cap and/or poly(A) tail dependency of translation initiation. The models predict that both cap and poly(A) tail dependencies of translation, and translatability of mRNAs are coupled with the structure of 5' UTR: the translation of mRNA with structured 5' UTR is strongly cap- and poly(A) tail-dependent; while translation of mRNA with unstructured 5' UTR is less cap- and poly(A) tail-dependent. We use these two models to explain: (1) the cap and poly(A) tail dependence of translation; (2) the effect of exogenous poly(A) on translation; (3) repression of host mRNA and translation of late adenovirus mRNA in the late phase of adenovirus infection; (4) repression of host mRNA and translation of Vaccinia virus mRNA in virus-infected cell; (5) heat shock repression of translation of normal mRNA and stimulation of translation of hsp mRNA; and (6) the synergistic effect of cap and poly(A) tail on stimulating translation. The kinetic proofreading scanning models provide a coherent interpretation of those phenomena.

5' Untranslated Regions↗

PubMed2004

Cleavage of poly(A)-binding protein by poliovirus 3C protease inhibits host cell translation: a novel mechanism for host translation shutoff.

Cleavage of eukaryotic translation initiation factor 4GI (eIF4GI) by viral 2A protease (2Apro) has been proposed to cause severe translation inhibition in poliovirus-infected cells. However, infections containing 1 mM guanidine-HCl result in eIF4GI cleavage but only partial translation shutoff, indicating eIF4GI cleavage is insufficient for drastic translation inhibition. Viral 3C protease (3Cpro) cleaves poly(A)-binding protein (PABP) and removes the C-terminal domain (CTD) that interacts with several translation factors. In HeLa cell translation extracts that exhibit cap-poly(A) synergy, partial cleavage of PABP by 3Cpro inhibited translation of endogenous mRNAs and reporter RNA as effectively as complete cleavage of eIF4GI and eIF4GII by 2Apro. 3Cpro-mediated translation inhibition was poly(A) dependent, and addition of PABP to extracts restored translation. Expression of 3Cpro in HeLa cells resulted in partial PABP cleavage and similar inhibition of translation. PABP cleavage did not affect eIF4GI-PABP interactions, and the results of kinetics experiments suggest that 3Cpro might inhibit late steps in translation or ribosome recycling. The data illustrate the importance of the CTD of PABP in poly(A)-dependent translation in mammalian cells. We propose that enteroviruses use a dual strategy for host translation shutoff, requiring cleavage of PABP by 3Cpro and of eIF4G by 2Apro.

3C Viral Proteases↗

PubMed2004

The pioneer translation initiation complex is functionally distinct from but structurally overlaps with the steady-state translation initiation complex.

The bulk of cellular proteins derive from the translation of eukaryotic translation initiation factor (eIF)4E-bound mRNA. However, recent studies of nonsense-mediated mRNA decay (NMD) indicate that cap-binding protein (CBP)80-bound mRNA, which is a precursor to eIF4E-bound mRNA, can also be translated during a pioneer round of translation. Here, we report that the pioneer round, which can be assessed by measuring NMD, is not inhibited by 4E-BP1, which is known to inhibit steady-state translation by competing with eIF4G for binding to eIF4E. Therefore, at least in this way, the pioneer round of translation is distinct from steady-state translation. eIF4GI, poly(A)-binding protein (PABP)1, eIF3, eIF4AI, and eIF2alpha coimmunopurify with both CBP80 and eIF4E, which suggests that each factor functions in both modes of translation. Consistent with roles for PABP1 and eIF2alpha in the pioneer round of translation, PABP-interacting protein 2, which is known to destabilize PABP1 binding to poly(A) and inhibit steady-state translation, as well as inactive eIF2alpha, which is also known to inhibit steady-state translation, also inhibit NMD. Polysome profiles indicate that CBP80-bound mRNAs are translated less efficiently than their eIF4E-bound counterparts.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Dominant negative mutants of mammalian translation initiation factor eIF-4A define a critical role for eIF-4F in cap-dependent and cap-independent initiation of translation.

Eukaryotic translation initiation factor-4A (eIF-4A) plays a critical role in binding of eukaryotic mRNAs to ribosomes. It has been biochemically characterized as an RNA-dependent ATPase and RNA helicase and is a prototype for a growing family of putative RNA helicases termed the DEAD box family. It is required for mRNA-ribosome binding both in its free form and as a subunit of the cap binding protein complex, eIF-4F. To gain further understanding into the mechanism of action of eIF-4A in mRNA-ribosome binding, defective eIF-4A mutants were tested for their abilities to function in a dominant negative manner in a rabbit reticulocyte translation system. Several mutants were demonstrated to be potent inhibitors of translation. Addition of mutant eIF-4A to a rabbit reticulocyte translation system strongly inhibited translation of all mRNAs studied including those translated by a cap-independent internal initiation mechanism. Addition of eIF-4A or eIF-4F relieved inhibition of translation, but eIF-4F was six times more effective than eIF-4A, whereas eIF-4B or other translation factors failed to relieve the inhibition. Kinetic experiments demonstrated that mutant eIF-4A is defective in recycling through eIF-4F, thus explaining the dramatic inhibition of translation. Mutant eIF-4A proteins also inhibited eIF-4F-dependent, but not eIF-4A-dependent RNA helicase activity. Taken together these results suggest that eIF-4A functions primarily as a subunit of eIF-4F, and that singular eIF-4A is required to recycle through the complex during translation. Surprisingly, eIF-4F, which binds to the cap structure, appears to be also required for the translation of naturally uncapped mRNAs.

Amino Acid Sequence↗

PubMed1999

Cotranslational formation of active photoprotein obelin in a cell-free translation system: direct ultrahigh sensitive measure of the translation course.

Translation of apoobelin mRNA in a cell-free wheat germ translation system in the presence of coelenterazine and molecular oxygen results in cotranslational formation of active photoprotein. Active obelin formation is recorded by its luminescence, either direct in the translation mixture in the presence of coelenterazine and calcium ions or in aliquots from the translation mixture. In the second case translation is carried out with coelenterazine and EGTA. Registration of the translation course by luminescence of the synthesized product in both cases allows use of apoobelin mRNA at very low concentrations as an internal marker for immediate measure of protein biosynthesis activity of in vitro translation systems. It is shown that the simultaneous translation of any other mRNA does not affect translation of photoprotein mRNAs under standard conditions. Continuous registration of luminescence in a cuvette of a liquid scintillation counter in photon-counting mode varies the time of signal accumulation in a wide temporal range, thus increasing the numerical values of the recorded signals. Registration of photoprotein luminescence during translation can be used to obtain additional information about the translation process, for example codon reading speed, about protein folding, and about the formation of active proteins on ribosomes.

Cell-Free System↗

PubMed1997

Translation-competent extracts from Saccharomyces cerevisiae: effects of L-A RNA, 5' cap, and 3' poly(A) tail on translational efficiency of mRNAs.

Yeast genetics has proven fruitful in the identification of key players that are involved in translational initiation. However, the exact roles of many translation initiation factors in translation initiation remain unknown. This has been due to lack of a suitable in vitro translation system in which the mode of action of certain translation factors can be studied. This report describes the preparation of cell-free Saccharomyces cerevisiae lysates that can mediate the translation of exogenously added mRNAs. Optimal translation required the absence of viral L-A RNA in the lysate and the presence of both a 5' cap and a 3' poly(A) tail on the mRNAs. A cooperative effect of cap and poly(A) tail on translation initiation was observed, a property that has been found to operate in intact yeast cells as well. In addition, the yeast lysates mediated translational initiation through several viral internal ribosome entry sites, demonstrating that the yeast translation apparatus can perform internal initiation. Thus, these lysates may be useful in the biochemical analysis of cap-dependent and cap-independent translation events.

Protein Biosynthesis↗