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Results for “translational efficiency”

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PubMed1993

Effect of tandem repeated AUG codons on translation efficiency of eukaryotic mRNA carrying a short leader sequence.

The effect on translation of multiple copies of the initiation codon AUG at the initiation site in a eukaryotic mRNA carrying a short leader sequence was tested in translation experiments in vitro. DNA, corresponding to a chimeric mRNA sequence consisting of the 5' leader region of brome mosaic virus (BMV) RNA4 and the goat pre-alpha-lactalbumin mRNA sequence, was prepared and transcribed in vitro using SP6 RNA polymerase. Site-directed mutagenesis was carried out to change the sequence around the initiation codon AUG. In a wheat germ translation system, the yield of protein obtained using the mRNA with a duplication of the AUG codons at the initiation site was 1.6 times that achieved when only one AUG was present. The rate of formation of the 80S initiation complex was measured by the ribosome binding assay using cycloheximide. A good correlation was observed between the ability to form the complex and translation efficiency.

Animals↗

PubMed2006

Effects of the Sabin-like mutations in domain V of the internal ribosome entry segment on translational efficiency of the Coxsackievirus B3.

The domain V within the internal ribosome entry segment (IRES) of poliovirus (PV) is expected to be important in its own neurovirulence because it contains an attenuating mutation in each of the Sabin vaccine strains. In this study, we try to find out if the results observed in the case of Sabin vaccine strains of PV can be extrapolated to another virus belonging to the same genus of enteroviruses but with a different tropism. To test this hypothesis, we used the coxsackievirus B3 (CVB3), known to be the most common causal agent of viral myocarditis. The introduction of the three PV Sabin-like mutations in the equivalent positions (nucleotides 484, 485, and 473) to the domain V of the CVB3 IRES results in significant reduced viral titer of the Sabin3-like mutant (Sab3-like) but not on those of Sab1- and Sab2-like mutants. This low titer was correlated with poor translation efficiency in vitro when all mutants were translated in rabbit reticulocyte lysates. However, elucidation by biochemical probing of the secondary structure of the entire domain V of the IRES of Sabin-like mutants reveals no distinct profiles in comparison with the wild-type counterpart. Prediction of secondary structure by MFOLD program indicates a structural perturbation of the stem containing the Sab3-like mutation, suggesting that specific protein-viral RNA interactions are disrupted, preventing efficient viral translation.

Animals↗

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↗

PubMed1994

Efficient translation of poly(A)-deficient mRNAs in Saccharomyces cerevisiae.

The polyadenylate tail of eukaryotic mRNAs is thought to influence various metabolic phenomena including mRNA stability, translation initiation, and nucleo-cytoplasmic transport. We have analyzed the fate of mRNAs following inactivation of poly(A) polymerase in Saccharomyces cerevisiae containing a temperature-sensitive, lethal mutation (pap1-1) in the gene for poly(A) polymerase (PAP1). Inactivation of poly(A) polymerase (Pap1) by shifting cells to the nonpermissive temperature resulted in the loss of at least 80% of measurable poly(A) within 60 min. Northern blot analysis revealed the disappearance of some mRNAs (CYH2 and HIS4) consistent with a role for poly(A) tails in mRNA stability. However, other mRNAs (TCM1, PAB1, ACT1, and HTB2) accumulate as poly(A)-deficient (A < approximately 25) transcripts as defined by an inability to bind oligo(dT)-cellulose. Sucrose density gradient analysis of polyribosomes revealed a twofold reduction in the amount of each size class of polyribosomes in shifted cells and a commensurate increase in free ribosomes. However, poly(A)-deficient mRNAs in shifted cells remain associated with the same size polyribosomes as poly(A)+ mRNAs in unshifted cells, indicating normal initiation of translation. RNase mapping of transcripts from pap1-1 cells revealed PAB1 mRNA to be poly(A)- whereas TCM1 exists as equal amounts of poly(A)- and poly(A)+ mRNA 60 min after shift. Interestingly, both of these classes of TCM1 mRNA appear in similar amounts in each polyribosome fraction indicating that ribosomes may not distinguish between them. These findings suggest that under conditions of excess translational capacity, poly(A)- and poly(A)+ mRNAs may initiate translation with comparable efficiencies.

Base Sequence↗

PubMed1990

Evidence for increased translational efficiency in the induction of P450IIE1 by solvents: analysis of P450IIE1 mRNA polyribosomal distribution.

The potential for enhanced translational processing of P450IIE1 mRNA during the early phase of P450IIE1 induction by pyridine or acetone was assessed by hybridization analysis of polyribosomal P450IIE1 mRNA distribution in rat hepatic tissue. Optical absorbance profiles of polyribosomal fractions exhibited an apparent shift at 5 h following pyridine administration relative to control. Slot and Northern blot analyses for P450IIE1 mRNA in the cytoplasmic extracts isolated from 5 h pyridine-treated rats demonstrated a shift in distribution of P450IIE1 message toward heavier polyribosomal fractions and Northern blot analysis suggested the presence of different populations of P450IIE1 mRNA. Slot blot analyses also demonstrated a shift in the polyribosomal distribution of P450IIE1 mRNA at 12 h following pyridine treatment; in contrast, hybridization analysis for P450IA1 revealed no shift in polyribosomal distribution of P450IA1 mRNA. Acute acetone administration to animals also resulted in a similar shift in polyribosomal distribution of P450IIE1 mRNA as compared to control. These data suggest that P450IIE1 mRNA shifts toward larger polyribosomes following acute exposure of animals to pyridine or acetone and provide evidence that induction of P450IIE1 at early times following acute pyridine or acetone administration involves enhanced translational efficiency through increased loading of ribosomes on P450IIE1 mRNA.

Acetone↗

PubMed1986

The translational efficiency of tRNA is a property of the anticodon arm.

We have reciprocally transplanted the anticodon arm sequences of a set of amber suppressor tRNA genes, using recombinant DNA techniques. By this means, a very efficient suppressor may be converted to a poor one, and the poorest tRNA to the efficiency of the best one. In tRNA molecules of normal 2 degrees and 3 degrees structure, the suppressor efficiencies of different composite tRNAs having the same anticodon arm sequence are approximately the same. Large numbers of simultaneous changes throughout the rest of the molecule do not affect the efficiency. Selective nucleotide modification as a result of varied anticodon arm sequences cannot explain these efficiencies. Efficiencies are also unlikely to differ because of selective aminoacylation. Measurement of in vivo tRNA shows, however, that tRNA levels do vary if the anticodon arm sequence is changed. If tRNA levels are normalized, the anticodon arm effect on the translational efficiency remains. Therefore, different anticodon arms, all of normal secondary structure, are not equivalent in translation. The most efficient sequences in this series resemble those found in natural tRNAs associated with similar anticodons, as is proposed in the extended anticodon theory (Yarus, M. (1982) Science 218, 646-652). These molecules also provide some information on the specificity of nucleotide modification enzymes and on determinants of the steady-state tRNA level.

Anticodon↗

PubMed1997

Co-operation of the 5' and 3' untranslated regions of ornithine decarboxylase mRNA and inhibitory role of its 3' untranslated region in regulating the translational efficiency of hybrid RNA species via cellular factor.

The 5' untranslated region (UTR) has an inhibitory role in the translatability of ornithine decarboxylase (ODC) mRNA and of hybrid mRNA species, whereas the ODC 3' UTR causes a partial release of this inhibition. We designed experiments to explore whether the co-operation between ODC 5' UTR and 3' UTR in the translational regulation is due to a direct interaction of those sequences or whether it is mediated by their interaction with cellular factor(s). We stably transfected Chinese hamster ovary (CHO)-K1 cells and transiently transfected COS-1 cells with expression vectors carrying different chimaeric DNAs having the luciferase (LUC) coding sequence as reporter gene, the ODC 5' UTR or the ODC 3' UTR, or both, in the appropriate positions. We compared the results obtained by assaying the LUC activities of both transfected cell lines with each chimaeric DNA with those observed by translating the hybrid RNAs in a translation system in vitro. When the ODC 3' UTR was present, we observed a partial release of the translation inhibition owing to the ODC 5' UTR only in vivo. The releasing effect was restored in vitro by the addition of cytoplasmic extracts from wild-type CHO-K1 or COS-1 cells, prepared 2 and 8 h after their release from serum starvation. We also observed a partial inhibition of the translatability of the hybrid RNA owing to the presence of the ODC 3' UTR itself; the translational efficiency could be rescued by cell extract from 8 h serum-stimulated cells. The co-operation between the ODC-UTRs might be mediated by factors expressed by cells during particular phases of the cell cycle. Excess copies of the ODC-UTRs, expressed in trans, could compete in binding limited amounts of such regulatory factors and remove them from interaction with the endogenous ODC mRNA. This phenomenon should be reflected by modifications of the kinetics of ODC and/or LUC activities during serum stimulation. The overexpression of the ODC 3' UTR determined an increase in both endogenous ODC activity and LUC activity. Moreover, in the transfectants expressing the hybrid RNA species bearing the ODC 3' UTR the basal ODC activity is higher than that observed in control cells. We suggest that excess copies of the ODC 3' UTR mis-regulate the endogenous ODC translatability, probably by tying up regulatory molecules expressed by cells in limited amounts and sequestering them from the ODC mRNA species they should interact with.

Animals↗

PubMed1982

Translational efficiency of transfer RNA's: uses of an extended anticodon.

Transfer RNA's are probably very strongly selected for translational efficiency. In this article, the argument is presented that the coding performance of the triplet anticodon is enhanced by selection of a matching anticodon loop and stem sequence. the anticodon plus these nearby sequence features (the extended anticodon) therefore contains more coding information than the anticodon alone and can perform more efficiently and accurately at the ribosome. This idea successfully accounts for the relative efficiencies of many transfer RNA's.

Base Sequence↗

PubMed1995

Picornavirus internal ribosome entry segments: comparison of translation efficiency and the requirements for optimal internal initiation of translation in vitro.

On the basis of primary sequence comparisons and secondary structure predictions, picornavirus internal ribosome entry segments (IRESes) have been divided into three groups (entero- and rhinoviruses; cardio- and and aphthoviruses; and hepatitis A virus). Here, we describe a detailed comparison of the ability of IRESes from each group to direct internal initiation of translation in vitro using a single dicistronic mRNA (the only variable being the IRES inserted into the dicistronic region). We studied the influence of various parameters on the capacity of six different picornaviral IRESes, and the non-picornaviral hepatitis C virus IRES, to direct internal initiation of translation: salt concentration, the addition of HeLa cell proteins to rabbit reticulocyte lysate translation reactions, the presence of foot-and-mouth disease virus Lb or human rhinovirus 2A proteinase. On the basis of the characteristics of IRES-driven translation in vitro, the picornaviral IRESes can be classified in a similar manner to when sequence homologies are considered. IRESes from each of the three groups responded differently to all of the parameters tested, indicating that while all of these elements can direct internal ribosome entry, the functional requirements for efficient IRES activity vary dramatically. In the individual optimal conditions for translation initiation, the best IRESes were those from the cardio- and aphthoviruses, followed by those from the enteroviruses, which exhibited up to 70% of the efficiency of the EMCV element in directing internal initiation of translation.

Base Sequence↗

PubMed2002

Post-transcriptional mechanisms control catalase synthesis during its light-induced turnover in rye leaves through the availability of the hemin cofactor and reversible changes of the translation efficiency of mRNA.

The enzyme catalase is light-sensitive. In leaves, losses caused by photoinactivation are replaced by new enzyme and the rate of de novo synthesis must be rapidly and flexibly attuned to fluctuating light conditions. In mature rye leaves, post-transcriptional mechanisms were shown to control the rate of catalase synthesis. The amount of the leaf catalase (CAT-1) transcript did not increase with light intensity, but was even higher after dark exposure of light-grown leaves. Initiation was apparently not limiting translation in the dark, as the association of the Cat1 mRNA with polysomes did not change notably under different light conditions. By analysing the translation of catalase polypeptides in cell-free systems with poly(A)+ RNA from leaves or with mRNA transcribed from a Cat1-containing cDNA clone, two mechanisms of post-transcriptional control were identified. First, translation of catalase depended on the presence of hemin. In leaves, the availability of hemin may signal the extent of catalase degradation as the hemin of the inactivated enzyme is recycled. Second, the translation efficiency of the Cat1 transcripts was reversibly modulated in a dose-dependent manner by the light intensity to which leaves were exposed, prior to extraction. The Cat1 mRNA from light-exposed leaves was translated much more efficiently than mRNA from dark-exposed leaves. The increase of its translation activity in vivo was not blocked by cordycepin but was suppressed by methylation inhibitors, indicating a reversible modification of pre-existing mRNA by methylation. Translation of in vitro synthesized Cat1 mRNA required a methylated cap (m7GpppG), but was virtually below detection when it contained an unmethylated cap (GpppG).

Catalase↗

PubMed2003

A survey of codon and amino acid frequency bias in microbial genomes focusing on translational efficiency.

Unequal use of synonymous codons has been found in several prokaryotic and eukaryotic genomes. This bias has been associated with translational efficiency. The prevalence of this bias across lineages is currently unknown. Here, a new method (GCB) to measure codon usage bias is presented. It uses an iterative approach for the determination of codon scores and allows the computation of an index of codon bias suitable for interspecies comparison. A server to calculate GCB-values of individual genes as well as a list of compiled results are available at www.g21.bio.uni-goettingen.de. The method was applied to complete bacterial genomes. The relation of codon usage bias with amino acid composition and the choice of stop codons were determined and discussed.

Amino Acids↗

PubMed1992

The influence of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo.

A method is described to determine simultaneously the effect of any changes in the ribosome-binding site (RBS) of mRNA on translational efficiency in Bacillus subtilis and Escherichia coli in vivo. The approach was used to analyse systematically the influence of spacing between the Shine-Dalgarno sequence and the initiation codon, the three different initiation codons, and RBS secondary structure on translational yields in the two organisms. Both B. subtilis and E. coli exhibited similar spacing optima of 7-9 nucleotides. However, B. subtilis translated messages with spacings shorter than optimal much less efficiently than E. coli. In both organisms, AUG was the preferred initiation codon by two- to threefold. In E. coli GUG was slightly better than UUG while in B. subtilis UUG was better than GUG. The degree of emphasis placed on initiation codon type, as measured by translational yield, was dependent on the strength of the Shine-Dalgarno interaction in both organisms. B. subtilis was also much less able to tolerate secondary structure in the RBS than E. coli. While significant differences were found between the two organisms in the effect of specific RBS elements on translation, other mRNA components in addition to those elements tested appear to be responsible, in part, for translational species specificity. The approach described provides a rapid and systematic means of elucidating such additional determinants.

Bacillus subtilis↗

PubMed2000

Post-transcriptional regulation of rat CYP2E1 expression: role of CYP2E1 mRNA untranslated regions in control of translational efficiency and message stability.

Altered expression of hepatic CYP2E1 by xenobiotic or physiological stimuli is largely mediated through post-transcriptional mechanisms that may include altered CYP2E1 mRNA translation and/or protein degradation. Examination of the polyribosomal distribution of rat hepatic P450 mRNAs indicated that, whereas nearly all of the CYP2B, CYP3A, and CYP4A mRNAs were recovered in the polysomal fractions, indicating active translation, approximately 30-40% of CYP2E1 mRNA was not associated with polysomes and therefore not actively engaged in protein synthesis. To examine the CYP2E1 mRNA molecule for sequences that might affect its translational efficiency, a series of CYP2E1 recombinant RNAs (rcRNAs) with modified 5' or 3' untranslated regions (UTRs) was translated in vitro using the rabbit reticulocyte lysate system. Deletion of most of the CYP2E1 5' UTR, which was predicted to contain secondary structure, increased in vitro CYP2E1 protein synthesis. Polysomal distribution analyses of 5'-modified rcRNAs demonstrated that, as seen for hepatic CYP2E1 mRNA, a substantial fraction of each CYP2E1 rcRNA was not associated with polysomes. The polysomal distribution analyses of the CYP2E1 rcRNAs also confirmed that the observed changes in CYP2E1 protein synthesis were associated with altered ribosomal loading. Deletion of the poly(A) tail, and partial or complete deletion of the 3' UTR, decreased CYP2E1 protein synthesis. These changes in protein synthesis were accompanied by increased degradation of the CYP2E1 rcRNAs. Incubation with translational inhibitors, but not increased levels of RNase inhibitor, decreased the degradation of the rcRNAs during in vitro translation. In conclusion, these studies suggest that secondary structure in the 5' UTR of CYP2E1 mRNA is at least partially responsible for the inefficient translation of this mRNA. The poly(A) tail and sequences contained within the 3' UTR appear to be important for protecting CYP2E1 mRNA from RNase activity associated with the translation machinery.

3' Untranslated Regions↗

PubMed2000

Efficient translation of rotavirus mRNA requires simultaneous interaction of NSP3 with the eukaryotic translation initiation factor eIF4G and the mRNA 3' end.

In contrast to the vast majority of cellular proteins, rotavirus proteins are translated from capped but nonpolyadenylated mRNAs. The viral nonstructural protein NSP3 specifically binds the 3'-end consensus sequence of viral mRNAs and interacts with the eukaryotic translation initiation factor eIF4G. Here we show that expression of NSP3 in mammalian cells allows the efficient translation of virus-like mRNA. A synergistic effect between the cap structure and the 3' end of rotavirus mRNA was observed in NSP3-expressing cells. The enhancement of viral mRNA translation by NSP3 was also observed in a rabbit reticulocyte lysate translation system supplemented with recombinant NSP3. The use of NSP3 mutants indicates that its RNA- and eIF4G-binding domains are both required to enhance the translation of viral mRNA. The results reported here show that NSP3 forms a link between viral mRNA and the cellular translation machinery and hence is a functional analogue of cellular poly(A)-binding protein.

Animals↗

PubMed2000

Comparative analysis of translation efficiencies of hepatitis C virus 5' untranslated regions among intraindividual quasispecies present in chronic infection: opposite behaviors depending on cell type.

Hepatitis C virus (HCV) RNA translation initiation is dependent on the presence of an internal ribosome entry site (IRES) that is found mostly in its 5' untranslated region (5' UTR). While exhibiting the most highly conserved sequence within the genome, the 5' UTR accumulates small differences, which may be of biological and clinical importance. In this study, using a bicistronic dual luciferase expression system, we have examined the sequence of 5' UTRs from quasispecies characterized in the serum of a patient chronically infected with HCV genotype 1a and its corresponding translational activity. Sequence heterogeneity between IRES elements led to important changes in their translation efficiency both in vitro and in different cell cultures lines, implying that interactions of RNA with related transacting factors may vary according to cell type. These data suggest that variants occasionally carried by the serum prior to reinfection could be selected toward different compartments of the same infected organism, thus favoring the hypothesis of HCV multiple tropism.

5' Untranslated Regions↗

PubMed2004

Translation efficiency mediated by the 5' untranslated region greatly affects protein production in Aspergillus oryzae.

We demonstrate that the 5' untranslated region (5'UTR) plays an important role in determining translation efficiency in Aspergillus oryzae, using a model beta-glucuronidase (GUS) expression system. Alterations in the 5' UTR resulted in an increase in GUS activity of up to eight-fold, without affecting mRNA levels. Moreover, using the most effective 5'UTR construct, we could achieve remarkable intracellular overproduction of GUS protein; and the GUS level reached more than 50% of the total soluble protein. This is the first experimental evidence indicating the feasibility of improving recombinant protein yield by promoting translation initiation in filamentous fungi.

5' Untranslated Regions↗

PubMed1973

Efficient translation of tobacco mosaic virus RNA and rabbit globin 9S RNA in a cell-free system from commercial wheat germ.

Extracts prepared from commercially available wheat germ efficiently translate messenger RNAs from either viral or eukaryotic origin. The addition of tobacco mosaic virus RNA stimulated amino-acid incorporation more than 100-times and rabbit globin 9S RNA between 20- and 30-times. The in vitro product directed by globin 9S RNA comigrated precisely with authentic rabbit globin on sodium dodecyl sulfate-polyacrylamide gels. Furthermore, during high voltage ionophoresis two [(35)S]methionine-labeled tryptic peptides synthesized in vitro comigrated in two dimensions with alphaT5 and betaT5 tryptic peptides from authentic [(35)S]methionine-labeled rabbit globin.

Amino Acids↗

PubMed1998

A common genetic polymorphism (46 C to T substitution) in the 5'-untranslated region of the coagulation factor XII gene is associated with low translation efficiency and decrease in plasma factor XII level.

We studied the Hga I polymorphism (46 C/T) in the 5'-untranslated region of the coagulation factor XII (FXII) gene corresponding to four bases upstream from the ATG translation initiation codon. By using allele-specific restriction analysis with restriction endonuclease Hga I, the allele frequency of 46C/T was estimated to be 0.27/0.73 in Orientals (allele number =152), and conversely, 0.8/0.2 in Caucasians (allele number =40). Because it has been reported that plasma levels of FXII were lower in Orientals than in Caucasians, we investigated the relationship between this polymorphism and plasma levels of FXII. As a result, there were significant differences in plasma FXII levels between these three allele types: C/C,170+/-38% (178+/-27%); C/T, 141+/-29% (123+/-34%); and T/T, 82+/-19% (61+/-11%) [FXII activity (FXII antigen levels)]. In heterozygotes of 46 C/T both alleles were equally transcribed in hepatocytes, as determined by reverse transcription polymerase chain reaction (RT-PCR), suggesting little influence of the polymorphism at the level of transcription or on the stability of mRNA. In in vitro transcription/translation analysis, less FXII was produced from cDNA containing 46 T than from that containing 46 C. Therefore, it is highly likely that the 46 T polymorphism in the FXII gene decreased the translation efficiency and led to low plasma levels of FXII activity and antigen, probably due to the creation of another ATG codon and/or impairment of the consensus sequence for the translation initiation scanning model.

Adolescent↗