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Results for “translational efficiency”

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PubMed1991

The cap and poly(A) tail function synergistically to regulate mRNA translational efficiency.

The cap structure and the poly(A) tail are important regulatory determinants in establishing the translational efficiency of a messenger RNA. Although the mechanism by which either determinant functions remains poorly characterized, the interaction between the poly(A) tail-poly(A)-binding protein complex and events occurring at the 5' terminus during translation initiation has been an intriguing possibility. In this report, the mutual dependence of the cap and the poly(A) tail was studied. Poly(A)+ and poly(A)- luciferase (Luc) mRNAs generated in vitro containing or lacking a cap were translated in vivo in tobacco protoplasts, Chinese hamster ovary cells, and yeast following delivery by electroporation. The poly(A) tail-mediated regulation of translational efficiency was wholly dependent on the cap for function. Moreover, cap function was enhanced over an order of magnitude by the presence of a poly(A) tail. The relative differences in stability between the mRNAs could not account for the synergism. The synergism between the cap and poly(A) tail was not observed in yeast cells in which active translation had been disrupted. In addition, the synergism was not observed in in vitro translation lysates. These data demonstrate that the cap and the poly(A) tail are interdependent for optimal function in vivo and suggest that communication between the two regulatory determinants may be important in establishing efficient translation.

Animals↗

PubMed2001

Codon and base biases after the initiation codon of the open reading frames in the Escherichia coli genome and their influence on the translation efficiency.

Nucleotide sequences around the boundaries of all open reading frames in the Escherichia coli whole genome were analyzed. Characteristic base biases were observed after the initiation codon and before the termination codon. We examined the effect of the base sequence after the initiation codon on the translation efficiency, by introducing mutations after the initiation codon of the E. coli dihydrofolate reductase (DHFR) gene, considering codon and base biases, and using in vitro and in vivo translation systems. In both assay systems, the two most frequent second codons, AAA and AAU, enhanced the translation efficiency compared with the wild type, whereas the effects of lower frequency codons were not significant. Experiments using 16S rRNA variants with mutations in the putative complementary sequence to the region downstream of the initiation codon showed that the translation efficiency of none of the DHFR mutants was affected. These results demonstrate that the statistically most frequent sequences for the second codon enhance translation efficiency, and this effect seems to be independent of base pairing between mRNA and 16S rRNA.

Bacterial Proteins↗

PubMed1995

Translation efficiency of the human T-cell leukemia virus (HTLV-2) gag gene modulates the frequency of ribosomal frameshifting.

The regulation of ribosomal frameshifting during translation of the polycistronic mRNA of human T-cell leukemia virus (HTLV) was studied in a cell-free system. Translation inhibitors such as cycloheximide and puromycin antibiotics were much more effective in blocking the synthesis of the frameshift polypeptide Gag-Pro than the synthesis of the Gag product. The preferential inhibition of the frameshift polypeptide Gag-Pro by the two antibiotics was not a reflection of the different sizes of the two gene products, but rather a consequence of the effect of the inhibitors on ribosomal translation efficiencies. To further analyze the effect of translation efficiencies on ribosomal frameshifting, we compared the translation of 5'-capped RNA to noncapped RNA. The translation of 5'-capped RNA was higher, as expected. Consequently, ribosomal frameshifting producing the Gag-Pro polypeptide was enhanced when compared to the translation of noncapped RNA. Taken together these results indicate that efficiencies of translation, in conjunction with the cis regulatory genetic elements at the frameshift sites, determine the ratio of the polypeptides Gag, Gag-Pro, and Gag-Pro-Pol produced in the HTLV-infected cell. Thus, physiological changes which affect the cellular translation machinery may alter the optimal ratio of these three polyprotein products needed for virus maturation.

Cell-Free System↗

PubMed2004

Identification of unique hepatitis C virus quasispecies in the central nervous system and comparative analysis of internal translational efficiency of brain, liver, and serum variants.

Reports of cerebral dysfunction in chronic hepatitis C virus (HCV) infection have led to the suggestion that HCV may infect the central nervous system (CNS). We used reverse transcription-PCR, cloning, and sequencing to define quasispecies for the HCV internal ribosomal entry site (IRES) and hypervariable region 1 (HVR1) in autopsy-derived brain, liver, lymph node, and serum samples. There was evidence of tissue compartmentalization of sequences in the brain in two patients, with between 24 and 55% of brain-derived IRES sequences absent from the serum, and significant phylogenetic and phenetic clustering of the brain and lymph node HVR1 sequences. The IRES initiates cap-independent translation of the viral polyprotein. Two unique brain-derived IRES mutations (C(204)-->A and G(243)-->A), which have previously been associated with lymphoid replication and altered translational efficiency in cell culture, were found in one patient. We used a dicistronic reporter vector to test whether brain-derived variants showed altered IRES-mediated translational efficiency, which might favor CNS infection. The translational efficiencies of the brain-derived IRES sequences were generally reduced compared to those of the master serum and liver sequences in rabbit reticulocyte cell lysates and two human cell lines, HuH7 (liver) and CHME3 (microglial). The C(204)-->A and G(243)-->A mutations showed preserved translational efficiency in HuH7 cells but reduced efficiency in CHME3 cells. Our data provide evidence that the CNS is a site of HCV replication, consistent with the recent demonstration of negative-strand HCV RNA in brain, and suggest that IRES polymorphisms may be important as a viral strategy of reduced translation to favor latency in the CNS.

Base Sequence↗

PubMed1985

Mutations upstream of the ribosome-binding site affect translational efficiency.

The DNA coding for the major outer membrane lipoprotein of Escherichia coli has been fused to the coding region of the beta-galactosidase gene to measure the effect of various mutations on the efficiency of translation initiation. The various mutants were made by either inserting or deleting a small number of nucleotides into or from a region just upstream of the ribosome-binding site. These small mutations dramatically affect translation initiation as measured by the production of beta-galactosidase. We postulate that these mutations affect translation initiation by altering the secondary structure of the messenger RNA. In one case, we predict that a stem and loop just upstream of the Shine-Dalgarno sequence sterically hinders the binding of the ribosome to the mRNA.

Base Sequence↗

PubMed1985

Microinjection of mRNA enhances translational efficiency of human adenovirus fiber message in monkey cells.

In monkey cells abortively infected with human adenovirus serotype 2, the synthesis of the fiber polypeptide of the virion capsid is reduced by at least a factor of 100 when compared with that in monkey cells productively infected with a host range mutant of adenovirus serotype 2 (Ad2hr400). However, the steady-state level of fiber-encoding mRNA present in abortively infected monkey cells is only reduced by a factor of 5 to 10. When mRNA isolated from abortively and productively infected monkey cells was microinjected into the cytoplasms of uninfected or abortively infected monkey cells, no differences in the efficiency of translation of the fiber messages from these two sources were observed. These results suggest that the block to synthesis of the fiber polypeptide in abortively infected monkey cells does not reside in the translational machinery of the abortively infected cells themselves but may involve compartmentalization of the fiber message within the cells or an altered processing of the fiber message which prevents correct presentation to the ribosomes.

Adenoviruses, Human↗

PubMed1983

Portable Shine-Dalgarno regions; nucleotides between the Shine-Dalgarno sequence and the start codon affect the translation efficiency.

This chapter describes a gene expression system of E. coli that contains a portable Shine-Dalgarno region. Transcription of this system is directed by a hybrid promoter derived from trp and lac-UV5 promoter sequences, which is followed by a region that encodes the portable Shine-Dalgarno region. We used a series of synthetic portable Shine-Dalgarno regions to vary the length (from 4 to 13 bases) of the Shine-Dalgarno by increasing the number of bases on the mRNA that are complementary to the 3' end of 16s rRNA. Increase in the Shine-Dalgarno region to 8 or 13 bases decreased the translation efficiency of the chimeric leukocyte interferon messenger by 40%. In another series of portable Shine-Dalgarno regions, we varied the four bases that follow the Shine-Dalgarno region. The presence of four A residues or four T residues in this region resulted in the highest translation efficiency. The presence of four C residues reduced translation efficiency by 50%, compared with A or T residues. The presence of four G residues after the Shine-Dalgarno region lowered translation efficiency by 75%, compared with A or T residues.

Cloning, Molecular↗

PubMed2005

In vitro and in vivo translational efficiencies of the 5' untranslated region from eight genotype 2 bovine viral diarrhea virus field isolates.

We determined the in vitro and in vivo translational efficiency mediated by the internal ribosomal entry site (IRES) from eight BVDV2 field isolates varying in virulence using a bicistronic reporter vector in rabbit reticulocyte lysates (RRL), and in primate and bovine cell lines. Using a T7-promoter system, the high virulence isolates had greater translational efficiencies in bovine lymphocytes (BL-3 cells), than did the low virulence isolates. The low virulence isolates translated with greater efficiencies than the high virulence isolates in RRL, African green monkey kidney (CV-1) and bovine turbinate (BT) cells. Our results demonstrate that despite a high degree of sequence identity in the 5' untranslated region (UTR), subtle differences in the primary and secondary structures, as well as differences in cell lines, influence translational efficiencies.

5' Untranslated Regions↗

PubMed2002

High translation efficiency is mediated by the encephalomyocarditis virus internal ribosomal entry sites if the natural sequence surrounding the eleventh AUG is retained.

Internal ribosomal entry sites (IRES) allow cap-independent translation and are sequences that are often used in gene therapy for strategies in which several genes need to be expressed. In this study, two different sequences of encephalomyocarditis virus (EMCV) IRES were compared for their translation efficiency in the context of retroviral vectors. When the sequence surrounding the 11th AUG of the IRES was conserved (IRESg), the translation efficiency was significantly higher than if the AUG of the downstream gene started with the 11th AUG of the IRES (IRESb). The translation efficiency with IRESg was influenced by the cell type and also by the nature of the transgene.

Animals↗

PubMed2002

A single nucleotide polymorphism at the splice donor site of the human MYH base excision repair genes results in reduced translation efficiency of its transcripts.

BACKGROUND: Adenine paired with 8-hydroxyguanine, a major oxidatively damaged DNA lesion, is excised by mutY homologue (MYH) base excision repair protein in human cells. Since genetic polymorphisms of DNA repair genes associated with the activities and the expression levels of their products may modulate cancer susceptibility of individuals, we investigated the effect of a single nucleotide polymorphism (SNP) in the MYH gene on the difference in the expression levels of its products. RESULTS: An aberrant size of the beta type nuclear form transcript was detected in a lung cancer cell line, VMRC-LCD, by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The transcript contained the intron 1 sequence, and it was due to alternative splicing resulting from IVS1+5G/C SNP. The presence of the upstream open reading frame (ORF) on the 5'-side of the native ORF in the beta type transcript from the IVS1+5C allele could reduce the translation efficiency of the transcript into the nuclear form protein. Thus, expression vectors bearing the 5'-untranslated region sequence of either the IVS1+5G or 5C allele were constructed. In vitro translation analysis, as well as Western blot and quantitative RT-PCR analyses of the H1299 lung cancer cell line transfected with these vectors, revealed that the translation efficiency of the IVS1+5C transcript into MYH protein was much lower (approximately 30) than that of the IVS1+5G transcript. CONCLUSIONS: The SNP at the splice donor site of the MYH gene resulted in reduced translation efficiency of its transcripts. This is the fourth case of single nucleotide variations that cause alterations in translation initiation sites and translation efficiencies in human cells.

Alleles↗

PubMed2005

Differences in the translation efficiency and mRNA stability mediated by 5'-UTR splice variants of human SP-A1 and SP-A2 genes.

Surfactant protein A (SP-A) plays an important role in host defense, modulation of inflammatory processes, and surfactant-related functions of the lung. The human SP-A (hSP-A) locus consists of two functional genes, SP-A1 and SP-A2. Several hSP-A 5'-untranslated region (UTR) splice variants for each gene have been characterized and shown to be translated in vitro and in vivo. In this report, we investigated the role of hSP-A 5'-UTR splice variants on SP-A production and molecular mechanisms involved. We used in vitro transient expression of hSP-A 5'-UTR constructs containing luciferase as the reporter gene and quantitative real-time PCR to study hSP-A 5'-UTR-mediated gene expression. We found that 1) the four (A'D', ABD, AB'D', and A'CD') 5'-UTR splice variants under study enhanced gene expression, by increasing luciferase activity from 2.5- to 19.5-fold and luciferase mRNA from 4.3- to 8.8-fold compared with the control vector that lacked hSP-A 5'-UTR; 2) all four 5'-UTR splice variants studied regulated mRNA stability. The ABD variant exhibited the lowest rate of mRNA decay compared with the other three constructs (A'D', AB'D', and A'CD'). These three constructs also exhibited significantly lower rate of mRNA decay compared with the control vector; 3) based on the indexes of translational efficiency (luciferase activity/mRNA), ABD and AB'D' exhibited higher translational efficiency compared with the control vector, whereas the translational efficiency of each A'D' and A'CD' was lower than that of the control vector. These findings indicate that the hSP-A 5'-UTR splice variants play an important role in both SP-A translation and mRNA stability.

5' Untranslated Regions↗

PubMed1996

Translational efficiency is regulated by the length of the 3' untranslated region.

All polyadenylated mRNAs contain sequence of variable length between the coding region and the poly(A) tail. Little has been done to establish what role the length of the 3' untranslated region (3'UTR) plays in posttranscriptional regulation. Using firefly luciferase (luc) reporter mRNA in transiently transfected Chinese hamster ovary (CHO) cells, we observed that the addition of a poly(A) tail increased expression 97-fold when the length of the 3'UTR was 19 bases but that its stimulatory effect was only 2.3-fold when the length of the 3'UTR was increased to 156 bases. The effect of the luc 3'UTR on poly(A) tail function was orientation independent, suggesting that its length and not its primary sequence was the important factor. Increasing the length of the 3'UTR increased expression from poly(A)- mRNA but had little effect on poly(A)+ mRNA. To examine the effect of length on translational efficiency and mRNA stability, a 20-base sequence was introduced and reiterated downstream of the luc stop codon to generate a nested set of constructs in which the length of the 3'UTR increased from 4 to 104 bases. For poly(A)- reporter mRNA, translational efficiency in CHO cells increased 38-fold as the length of the 3'UTR increased from 4 to 104 bases. Increasing the length of the 3'UTR beyond 104 bases increased expression even further. Increasing the length of the 3'UTR also resulted in a 2.5-fold stabilization of the reporter mRNA. For poly(A)+ mRNA, the translational efficiency and mRNA half-life increased only marginally as the length of the 3'UTR increased from 27 to 161 bases. However, positioning the poly(A) tail only 7 bases downstream of the stop codon resulted in a 39-fold reduction in the rate of translation relative to a construct with a 27-base 3'UTR, which may be a consequence of the poly(A) tail-poly(A)-binding protein complex functioning as a steric block to translocating ribosomes as they approached the termination codon. The optimal length of the 3' noncoding region for maximal poly(A) tail-mediated stimulation of translation is approximately 27 bases. These data suggest that the length of the 3'UTR plays an important role in determining both the translational efficiency and the stability of an mRNA.

Animals↗

PubMed2001

The initiation codon affects ribosome binding and translational efficiency in Escherichia coli of cI mRNA with or without the 5' untranslated leader.

Translational efficiency of an AUG, CUG, GUG, or UUG initiation codon was measured for the naturally leaderless cI mRNA from bacteriophage lambda. In a cI-lacZ translational fusion, only AUG supported a high level of expression; GUG supported a low level of expression, while UUG and CUG expression was barely above background levels. Addition of an untranslated lac leader and Shine-Dalgarno sequence to cI increased expression but still showed a dependence on an AUG for maximum expression. cI-lacZ mRNA with an AUG initiation codon showed a greater in vitro ribosome binding strength and a higher level of full-length in vivo mRNA, suggesting that the initiation codon is an important determinant of ribosome binding strength and translational efficiency for mRNA with or without the 5' untranslated leader.

5' Untranslated Regions↗

PubMed1986

Alpha-thalassemia due to the deletion of nucleotides -2 and -3 preceding the AUG initiation codon affects translation efficiency both in vitro and in vivo.

We previously hypothesized that a 2 nucleotide deletion, causing a A-greater than C change at position -3 preceding the ATG initiation codon of alpha globin gene, reduced translation efficiency of alpha globin mRNA and was responsible for a form of alpha + thalassemia displayed by an Algerian patient. We presently show that this deletion leads to a 30-45% reduction in translation efficiency of synthetic alpha globin mRNA in rabbit reticulocyte lysate. In other experiments, we constructed alpha/G gamma hybrid globin genes in which the 3' end of normal or mutated alpha globin genes downstream to the ATG initiation codon was substituted by the 3' part of a G gamma globin gene. COS cells transfected with either of these 2 hybrid genes were shown to synthesize a similar amount of alpha/G gamma hybrid mRNAs but 50% less G gamma globin when transfected with the alpha/G gamma hybrid gene carrying the deletion. These results definitively establish that the 2 nucleotide deletion reduces translation efficiency by 30-50%. This contrasts with the 93% reduction induced by a similar A-greater than C change at position -3 in the different nucleotide context preceding the ATG codon of the rat preproinsulin gene.

Base Sequence↗

PubMed2001

Different Na, K-ATPase mRNA(beta1) species exhibit unique translational efficiencies.

We have previously identified five Na, K-ATPase beta1-mRNA species that are expressed in the rat heart, kidney, and brain. These mRNAs which are unequal in their abundance have an identical coding region but differ in the length of their 5'- and 3'-untranslated regions (UTRs). In this study we examined the possibility that the beta1-mRNA species exhibit differential translational efficiencies. We constructed expression plasmids encoding each of the five mRNAs and transcribed and translated them in vitro. Using rabbit reticulocyte system we determined the translation of the different mRNAs under conditions optimized for each beta1-cRNA and under an equivalent (standard) condition. The longest beta1-cRNA species (initiating at the first transcription start site and ending at the last [fifth] poly(A) site) exhibited the lowest relative translational efficiency averaging 0.2 +/- 0.05 units/mol of cRNA compared to the shortest beta1-cRNA species initiating at the first transcription start site and ending at the first poly(A) signal (with an assigned relative value of 1.0). These results suggested that the different translation rates of beta1-mRNAs may be due to their 3'-UTRs. To further define the role of beta1-3'-UTR, chimeric luciferase constructs containing different segments of the beta1-3'-UTR were transiently transfected into Clone 9 cells. Compared to the chimeric construct containing the shortest beta1-3'-UTR segment (ending at the first poly(A) site), the construct containing the full-length beta1-3'-UTR exhibited a luciferase expression of 0.23 +/- 0.04. To control for potential changes in the abundance of the expressed chimeric mRNAs which may lead to differences in luciferase expression, luciferase activity was normalized against chimeric luciferase-mRNA content measured in mixtures of cells stably transfected with the above constructs. The ratio of luciferase activity/chimeric luciferase-mRNA content in cells expressing the construct containing the entire beta1-3'-UTR region was 0.17 that in cells expressing chimeric luciferase mRNA containing beta1-3'-UTR up to the first poly(A) signal (P < 0.05). We conclude that the translational efficiency of the different beta1-mRNA species is negatively regulated by the 3'-UTR of the mRNA and that a regulating region appears to be localized between the second and fifth poly(A) signals of beta1-mRNA.

3' Untranslated Regions↗

PubMed1995

The first and third uORFs in RSV leader RNA are efficiently translated: implications for translational regulation and viral RNA packaging.

Rous sarcoma virus (RSV) RNA leader contains three short upstream open reading frames. We have shown recently that both uORFs 1 and 3 influence in vivo translation of the downstream gag gene and are involved in the virus RNA packaging process. In this report, we have studied the translational events occurring at the upstream AUGs in vivo. We show that (i) the first and third AUGs are efficient translational initiation sites; (ii) ribosomes reinitiate efficiently at AUG3; and (iii) deletions in the intercistronic distance between uORF1 and 3 (which is well conserved among avian strains) prevent ribosome initiation at AUG3, thus increasing translation efficiency at the downstream AUGgag. The roles of the uORFs in translation and packaging are discussed.

Animals↗

PubMed1986

Multiple cis-acting elements modulate the translational efficiency of GCN4 mRNA in yeast.

The expression of the GCN4 gene in yeast is regulated at the translational level: growth of yeast cells under amino acid starvation conditions results in an increase in the translational efficiency of GCN4 mRNA. A sequence within the 5' untranslated region of this mRNA, which contains four small open reading frames, acts in cis to suppress translation when growth occurs in rich media. In this report, we have analyzed the effects on translation of a series of deletion, insertion, and substitution mutations in the 5' untranslated region of GCN4 mRNA. This analysis showed that at least two distinct cis elements located within the region of the small upstream open reading frames are required, in conjunction with trans positive elements, for the translational activation of GCN4 mRNA. We propose that the translational efficiency of GCN4 mRNA is modulated by the rate of translation initiation at the upstream AUG codons.

Base Sequence↗

PubMed2001

Selenium regulation of transcript abundance and translational efficiency of glutathione peroxidase-1 and -4 in rat liver.

Glutathione peroxidase (GPX)1 mRNA in rat liver falls dramatically during Se deficiency to levels that are approx. 10% of Se-adequate levels. This regulation is mediated by mRNA stability, and is hypothesized to involve nonsense-mediated mRNA decay. mRNA levels for GPX4 and other selenoproteins are much less regulated by Se status. To evaluate the relative contribution of mRNA abundance versus translational efficiency to overall regulation of GPX1 expression, we quantified GPX1, GPX4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts per cell in rat liver. Surprisingly, we found that GPX1 transcripts in Se deficiency are moderately abundant and similar in abundance to GAPDH and other selenoprotein mRNAs; Se supplementation increases GPX1 mRNA so that it is 30-fold higher than GAPDH mRNA. Translational efficiency of GPX1 mRNA is half of that of GPX4. Translational efficiency of GPX1 mRNA increases approx. 20-fold with Se supplementation and appears to switch GPX1 mRNA from nonsense-mediated degradation to translation. This regulatory switch can explain why GPX1 expression is an excellent parameter for assessment of Se status.

Animals↗