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PubMed2000

Structure and function of a cap-independent translation element that functions in either the 3' or the 5' untranslated region.

Barley yellow dwarf virus RNA lacks both a 5' cap and a poly(A) tail, yet it is translated efficiently. It contains a cap-independent translation element (TE), located in the 3' UTR, that confers efficient translation initiation at the AUG closest to the 5' end of the mRNA. We propose that the TE must both recruit ribosomes and facilitate 3'-5' communication. To dissect its function, we determined the secondary structure of the TE and roles of domains within it. Nuclease probing and structure-directed mutagenesis revealed that the 105-nt TE (TE105) forms a cruciform secondary structure containing four helices connected by single-stranded regions. TE105 can function in either UTR in wheat germ translation extracts. A longer viral sequence (at most 869 nt) is required for full cap-independent translation in plant cells. However, substantial translation of uncapped mRNAs can be obtained in plant cells with TE105 combined with a poly(A) tail. All secondary structural elements and most primary sequences that were mutated are required for cap-independent translation in the 3' and 5' UTR contexts. A seven-base loop sequence was needed only in the 3' UTR context. Thus, this loop sequence may be involved only in communication between the UTRs and not directly in recruiting translational machinery. This structural and functional analysis provides a framework for understanding an emerging class of cap-independent translation elements distinguished by their location in the 3' UTR.

3' Untranslated Regions↗

PubMed2001

A somatic mutation in the 5'UTR of BRCA1 gene in sporadic breast cancer causes down-modulation of translation efficiency.

Mutations in the 5' UTR which cause increment/decrement of translation efficiency have been recently described as a novel molecular mechanism of disease. Alterations in the consensus sequence for the translation initiation may promote context-dependent leaky scanning of ribosomes and/or initiation from a downstream AUG codon. Initiation of translation from a downstream in-frame AUG codon in BRCA1 gene was recently identified in normal cells and possibly in breast cancer. Here we present further insight into BRCA1 translational pathophysiology investigating the role of the canonical structure of the initiation consensus sequence of BRCA1. We have analysed the effect of a somatic point mutation (117 G>C) in position -3 with respect to the AUG of the BRCA1 gene, identified in a highly aggressive sporadic breast cancer. We constructed chimeric genes encoding the luciferase reporter sequence downstream of the wild type or the mutated BRCA1 5'UTR. These transcripts were tested for their activity in in vitro and in vivo systems. In in vitro transcription/translation assays the estimated translation efficiency of the construct with the mutated BRCA1 5'UTR was 30-50% lower than that with the wild type BRCA1 5'UTR. The same chimeric genes were analysed for their expression in vivo by transient transfection in human cells. While the two constructs were equally transcribed, the plasmid carrying the mutated sequence produced 70% less luciferase activity compared to the wild type sequence. Finally, to obtain a direct evaluation on translational efficiency in vivo, we analysed mRNA translation on translationally active and non-active ribosomes separated from transfected cells. Mutant mRNA was partially localized in subpolysomal particles analytically confirming a polysome recruitment defect. Thus, characterization of BRCA1 5'UTR and translation efficiency seems to provide new insight into BRCA1 role in breast and ovarian cancer pathogenesis.

5' Untranslated Regions↗

PubMed1996

Non-canonical translation mechanisms in plants: efficient in vitro and in planta initiation at AUU codons of the tobacco mosaic virus enhancer sequence.

The 5' untranslated leader (Omega sequence) of tobacco mosaic virus (TMV) genomic RNA was utilized as a translational enhancer sequence in expression of the 17 kDa putative movement protein (pr17) of potato leaf roll luteovirus (PLRV). In vitro translation of RNAs transcribed from appropriate chimeric constructs, as well as their expression in transgenic potato plants, resulted in the expected wild-type pr17 protein, as well as in larger translational products recognized by pr17-specific antisera. Mutational analyses revealed that the extra proteins were translated by non-canonical initiation at AUU codons present in the wild-type Omega sequence. In the plant system translation initiated predominantly at the AUU codon at positions 63-65 of the Omega sequence. Additional AUU codons in a different reading frame of the Omega sequence also showed the capacity for efficient translation initiation in vitro. These results extend the previously noted activity of the TMV 5' leader sequence in ribosome binding and translation enhancement in that the TMV translation enhancer can mediate non-canonical translation initiation in vitro and in vivo.

Base Sequence↗

PubMed1990

Translation of cucumber necrosis virus RNA in vitro.

The in vitro translation products directed by cucumber necrosis virus (CNV) RNA were analysed in both rabbit reticulocyte lysate and wheatgerm extract cell-free translation systems. In rabbit reticulocyte lysates, one major protein of approximate Mr 34.6K was produced. In wheatgerm extracts, four proteins of approximate Mr values 41.6K, 34.6K, 24K and 20K were produced. The genomic locations of the CNV in vitro translation products were determined using several experimental approaches including, first, hybrid-arrested translation using negative-sense RNA corresponding to selected regions of the CNV genome, second, in vitro translation of synthetic positive-sense CNV transcripts and third, in vitro translation of CNV virion RNA fractionated according to size. Together these experiments demonstrated that the protein of Mr 34.6K is derived from the 5'-proximal coding region, the 41.6K protein is derived from an internal coding region, and that at least one but probably both the 24K and 20K proteins are derived from the 3'-terminal coding region. In addition, immunoprecipitation of in vitro translation products using anti-CNV polyclonal serum demonstrated that the 41.6K protein is the coat protein. The templates for the expression of CNV cistrons were investigated by in vitro translation of sucrose gradient-fractionated CNV virion RNA as well as in vitro translation of positive-sense synthetic transcripts.

Blotting, Northern↗

PubMed1999

Ribosomal pausing and scanning arrest as mechanisms of translational regulation from cap-distal iron-responsive elements.

Iron regulatory protein 1 (IRP-1) binding to an iron-responsive element (IRE) located close to the cap structure of mRNAs represses translation by precluding the recruitment of the small ribosomal subunit to these mRNAs. This mechanism is position dependent; reporter mRNAs bearing IREs located further downstream exhibit diminished translational control in transfected mammalian cells. To investigate the underlying mechanism, we have recapitulated this position effect in a rabbit reticulocyte cell-free translation system. We show that the recruitment of the 43S preinitiation complex to the mRNA is unaffected when IRP-1 is bound to a cap-distal IRE. Following 43S complex recruitment, the translation initiation apparatus appears to stall, before linearly progressing to the initiation codon. The slow passive dissociation rate of IRP-1 from the cap-distal IRE suggests that the mammalian translation apparatus plays an active role in overcoming the cap-distal IRE-IRP-1 complex. In contrast, cap-distal IRE-IRP-1 complexes efficiently repress translation in wheat germ and yeast translation extracts. Since inhibition occurs subsequent to 43S complex recruitment, an efficient arrest of productive scanning may represent a second mechanism by which RNA-protein interactions within the 5' untranslated region of an mRNA can regulate translation. In contrast to initiating ribosomes, elongating ribosomes from mammal, plant, and yeast cells are unaffected by IRE-IRP-1 complexes positioned within the open reading frame. These data shed light on a characteristic aspect of the IRE-IRP regulatory system and uncover properties of the initiation and elongation translation apparatus of eukaryotic cells.

Animals↗

PubMed1996

[Translation of titles into English in Medicina Clínica: quality and influence of the Spanish language].

BACKGROUND: Journals that are not published solely in English have the titles of papers translated into English, the international language of medicine. The aim of this paper is to analyse the accuracy and quality of such translations in Medicina Clínica and to assess the influence of the morphology and syntax of Spanish on the English versions of the titles. METHODS: Two professional medical translators, one Spanish and the other English, each with a knowledge of both languages, compared the original Spanish and the English translations of the titles of the 292 papers and communications published in the 20 issues of volume 100 of Medicina Clínica. The discrepancies or "errors" were classified in five groups of increasing seriousness. RESULTS: Of the titles studied, 77% contained some sort of error (458 errors were detected). In 100 titles (34%) there were differences in meaning between the original Spanish and the English translations. Another 72 titles contained serious orthographical, lexical or grammatical mistakes, though the basic meaning was not distorted. Approximately a third of the lexical and grammatical errors were attributable to the direct influence of Spanish. CONCLUSIONS: The English translations of titles in Medicina Clínica contain numerous orthographical, lexical and gammatical mistakes. Serious errors of meaning in a number of translated titles could result in misinterpretation by readers who do not know Spanish. We recommend that the authors should play a part in the translation of the titles, as this should provide a simple and effective mean of improving the accuracy of the translations. Our comparison yielded much worse results than had been expected, which suggests that similar studies with other medical journals in Spanish and other languages would be justified.

Clinical Medicine↗

PubMed2024

An Intrinsically Disordered RNA Binding Protein Modulates mRNA Translation and Storage.

Proteins with intrinsically disordered regions (IDR) play diverse functions in regulating gene expression in the cell. Many of these proteins interact with cytoplasmic ribosomes. However, the molecular functions related to the interactions are largely unclear. In this study, using an abundant RNA-binding protein, Sbp1, with a structurally well-defined RNA recognition motif and an intrinsically disordered RGG domain as a model system, we investigated how an RNA binding protein with IDR modulates mRNA storage and translation. Using genomic and molecular approaches, we show that Sbp1 slows ribosome movement on cellular mRNAs and promotes polysome stacking or aggregation. Sbp1-associated polysomes display a ring-shaped structure in addition to a beads-on-string morphology visualized under the electron microscope, likely to be an intermediate slow translation state between actively translating polysomes and the translation-sequestered RNA granule. Moreover, the binding of Sbp1 to the 5'UTRs of mRNAs represses both cap-dependent and cap-independent translation initiation of proteins, many are functionally important for general protein synthesis in the cell. Finally, post-translational modifications at the arginine in the RGG motif change the Sbp1 protein interactome and play important roles in directing cellular mRNAs to either translation or storage. Taken together, our study demonstrates that under physiological conditions, intrinsically disordered RNA binding proteins promote polysome aggregation and regulate mRNA translation and storage using multiple distinctive mechanisms. This research also establishes a framework with which functions of other IDR-containing proteins can be investigated and defined.

RNA-Binding Proteins↗

PubMed2001

Translational control of the proteome: relevance to cancer.

Translational control is an important but relatively unappreciated mechanism that regulates levels of protein products. In addition to a global translational control that regulates the cell's response to external stimuli such as growth factors, cytokines, stress and viral infections, selective translational control has recently been demonstrated to affect many genes related to growth and apoptotic processes. Modifications in the 5'untranslated region of these specific mRNAs may lead to an up-regulation of the protein product by as much as 100-fold. Translational infidelity has been reported in some human cancers for oncogenes such as c-myc and mdm2. Furthermore, modulation of selective translational control has also been demonstrated in cells over-expressing the translation initiation factor elF4E. Elevated levels of elF4E were found in a broad spectrum of solid tumors (breast, head and neck, colon and bladder carcinomas as well as in non-Hodgkin's lymphomas). Other translation initiation factors and translation components such as elongation factors and ribosomal proteins have also been reported to be overexpressed in some human tumors. This review discusses the relevance of these observations to a cell's proteome and for tumorigenesis and how the genomics and proteomics can be used to advance our understanding of the role of translational control in cancer.

Eukaryotic Initiation Factor-4E↗

PubMed1984

Escherichia coli ribosomes translate in vivo with variable rate.

The question of whether or not 'rare' codons are translated with the same rate as 'common' codons was investigated by measuring the translation time for two genes, lacI and bla, rich in rare codons, and comparing the results with the translation times measured on fus, tsf, tuf and rpsA which have very few rare codons. The rate of synthesis of the lac repressor was first measured with the up-promoter mutation lacIq1 present on the high copy number plasmid pBR322. In such a strain the average translation times for lacI and bla were 50% slower than the rate calculated from the translation time for the four ribosomal proteins. In a strain having lacIq1 on an F'lac episome this difference was much smaller, thus slow translation of genes rich in rare codons is exaggerated in strains with increased drain on the rare codon tRNAs. The data do not exclude that only a subset of the rare codons is translated more slowly. Translation times were also measured in cells growing in different media, and the translation chain growth rate was found to increase by approximately 40% going from acetate medium to a fully supplemented medium.

Codon↗

PubMed1988

Proteins encoded by a complex chloroplast transcription unit are each translated from both monocistronic and polycistronic mRNAs.

Chloroplast genes are typically organized into polycistronic transcription units that give rise to complex sets of overlapping RNAs through a series of processing steps. The functional significance of this complicated mode of expression is unknown. To determine whether processing of the primary transcript is required to create translatable mRNAs, the translational properties of the RNAs derived from the maize psbB gene cluster (containing the psbB, psbH, petB and petD genes) were examined. Almost all of the approximately 20 RNAs derived from this region co-sediment with polysomes in sucrose gradients, suggesting that at least one coding region on most transcripts is translated. To determine which sequences are translated on each polycistronic RNA, antibodies to psbB, petB or petD proteins were used to immunoselect polysomes engaged in the synthesis of each protein. Northern and S1 nuclease analyses of the immunoselected RNAs revealed that (i) potential start codons within the petB and petD introns are not functional in translation; (ii) all transcripts containing spliced petB or petD sequences are translated to give these proteins, regardless of upstream or downstream sequences; (iii) psbB is translated from all transcripts encoding it. It is concluded that intercistronic processing is not required for translation of these RNAs, although certain processing steps may enhance translational efficiency.

Blotting, Northern↗

PubMed1994

Mechanism of post-segregational killing: translation of Hok, SrnB and Pnd mRNAs of plasmids R1, F and R483 is activated by 3'-end processing.

The gene systems hok/sok of R1, srnB of F and pnd of R483 mediate plasmid maintenance by killing of plasmid-free segregants. Translation of the very stable mRNAs encoding the killer proteins is regulated by small unstable antisense RNAs. The differential decay rates of the inhibitory antisense RNAs and the mRNAs encoding the killer proteins is the basis for the onset of killer mRNA translation in newborn plasmid-free segregants and the killing of these cells. We have suggested previously that this requires that the killer mRNAs occur in two forms. A translationally inactive form was proposed to be converted into a 3'-truncated, translationally active mRNA. In the presence of the antisense RNA, translation from this killer mRNA should be inhibited. In this communication we present in vivo and in vitro evidence that support this model. The requirement for 3'-processing for killer gene expression is demonstrated. By using in vitro techniques it is shown that full-length Hok mRNA is translationally inactive, whereas a 3'-end truncated version of the Hok mRNA is translationally active. In vitro secondary structure probing suggests that the 3'-end of the full-length Hok mRNA folds back onto the translational initiation region of the mok gene and thereby inhibits translation of the mRNA. By inference we conclude that the Pnd and SrnB mRNAs are regulated by a similar mechanism.

Aminoglycosides↗

PubMed1995

Repression of cap-dependent translation by 4E-binding protein 1: competition with p220 for binding to eukaryotic initiation factor-4E.

An important aspect of the regulation of gene expression is the modulation of translation rates in response to growth factors, hormones and mitogens. Most of this control is at the level of translation initiation. Recent studies have implicated the MAP kinase pathway in the regulation of translation by insulin and growth factors. MAP kinase phosphorylates a repressor of translation initiation [4E-binding protein (BP) 1] that binds to the mRNA 5' cap binding protein eukaryotic initiation factor (eIF)-4E and inhibits cap-dependent translation. Phosphorylation of the repressor decreases its affinity for eIF-4E, and thus relieves translational inhibition. eIF-4E forms a complex with two other polypeptides, eIF-4A and p220, that promote 40S ribosome binding to mRNA. Here, we have studied the mechanism by which 4E-BP1 inhibits translation. We show that 4E-BP1 inhibits 48S pre-initiation complex formation. Furthermore, we demonstrate that 4E-BP1 competes with p220 for binding to eIF-4E. Mutants of 4E-BP1 that are deficient in their binding to eIF-4E do not inhibit the interaction between p220 and eIF-4E, and do not repress translation. Thus, translational control by growth factors, insulin and mitogens is affected by changes in the relative affinities of 4E-BP1 and p220 for eIF-4E.

Adaptor Proteins, Signal Transducing↗

PubMed2003

IRES-dependent translational control of Cbfa1/Runx2 expression.

The P1 and P2 promoters of the Cbfa1/Runx2 gene produce Type I and II mRNAs with distinct complex 5'-untranslated regions, respectively designated UTR1 and UTR2. To evaluate whether the 5'-UTRs impart different translational efficiencies to the two isoforms, we created SV40 promoter-UTR-luciferase reporter (luc) constructs in which the translational potential of the 5'-UTR regions was assessed indirectly by measurement of luciferase activity in transfected cell lines in vitro. In MC3T3-E1 pre-osteoblasts, UTR2 was translated approximately twice as efficiently as the splice variants of UTR1, whereas translation of unspliced UTR1 was repressed. To determine if the UTRs conferred internal ribosome entry site (IRES)-dependent translation, we tested bicistronic SV40 promoter-Rluc-UTR-Fluc constructs in which Fluc is expressed only if the intercistronic UTR permits IRES-mediated translation. Transfection of bicistronic constructs into MC3T3-E1 osteoblasts demonstrated that both UTR2 and the spliced forms of UTR1 possess IRES activity. Similar to other cellular IRESs, activity increased with genotoxic stress induced by mitomycin C. In addition, we observed an osteoblastic maturation-dependent increase in IRES-mediated translation of both UTR2 and the spliced forms of UTR1. These findings suggest that Cbfa1 UTRs have IRES-dependent translational activities that may permit continued Cbfa1 expression under conditions that are not optimal for cap-dependent translation.

5' Untranslated Regions↗

PubMed2006

Expression of RUNX2 isoforms: involvement of cap-dependent and cap-independent mechanisms of translation.

RUNX2, a major regulator of skeletogenesis, is expressed as type-I and type-II isoforms. Whereas most eukaryotic mRNAs are translated by the cap-dependent scanning mechanism, translation of many mRNAs including type-I and type-II RUNX2 mRNAs has been reported to be initiated by a cap independent internal ribosomal entry site (IRES). Since the dicistronic plasmid assay used to demonstrate IRES has been questioned, we investigated the presence of IRES in RUNX2 mRNAs using dicistronic plasmid and mRNA assays. Our results show that the dicistronic plasmid assay cannot be used to demonstrate IRES in RUNX2 mRNAs because the intercistronic region of dicistronic plasmids containing the 5'-UTRs of both RUNX2 mRNAs operates as a cryptic promoter. In dicistronic mRNA transfection studies the 5'-UTRs of both RUNX2 mRNAs exhibited no IRES activity. When transfected into osteoblastic cells, monocistronic reporter mRNA preceded by the 5'-UTR of type-II RUNX2 (Type-II-FLuc-A100) was translated to a high degree only in the presence of a functional cap (m(7)GpppG); in contrast, luciferase mRNA preceded by the 5'-UTR of type-I RUNX2 mRNA (Type-I-FLuc-A100) was translated poorly in the presence of either m(7)GpppG or a nonfunctional cap (ApppG). Notably, in transfected cells inhibitors of cap-dependent translation suppressed the translation of m(7)GpppG-capped Type-II-FLuc-A100, but not ApppG-capped reporter mRNA preceded by the IRES-containing hepatitis C virus (HCV) 5'-UTR. Our study demonstrates that type-II RUNX2 mRNA is translated by the cap-dependent mechanism. Although efficient translation of type-I RUNX2 mRNA appears to require a process other than cap-dependent, the mechanism of type-I RUNX2 mRNA translation remains to be resolved.

5' Untranslated Regions↗

PubMed2007

Premature translation of transition protein 2 mRNA causes sperm abnormalities and male infertility.

During mammalian spermiogenesis somatic histones are replaced at first by transition proteins, which are in turn replaced by the protamines, forming the sperm nucleoprotamines. It is believed that transition protein 2 (Tnp2) is necessary for maintaining the normal processing of protamines and, consequently, the completion of chromatin condensation. The transition protein mRNAs are stored in translationally inert messenger ribonucleoprotein particles for up to 7 days until translational activation in elongated spermatids. Substantial evidence suggests an involvement of 3'untranslated region (UTR) in the translational regulation of the Tnp2 mRNAs. In order to determine the role of Tnp2 3'UTR in translational regulation and to study whether the translational repression of Tnp2 mRNA is necessary for normal spermatid differentiation in mice, we generated transgenic mice that carry a Tnp2-hGH transgene. In this transgene, 3'UTR of Tnp2 gene was replaced by 3' 3'UTR of human growth hormone gene. In these transgenic animals, transcription and translation of Tnp2 occur simultaneously in round spermatids which is an evidence for involvement of Tnp2 3'UTR in its translation repression. Premature translation of Tnp2 mRNA caused abnormal head morphogenesis, reduced sperm motility and male infertility. These results show clearly that a strict temporal and stage-specific Tnp2 translation is necessary for the correct differentiation of round spermatids into mature spermatozoa and for male fertility.

3' Untranslated Regions↗

PubMed1996

Ribosome-mediated translational pause and protein domain organization.

Because regions on the messenger ribonucleic acid differ in the rate at which they are translated by the ribosome and because proteins can fold cotranslationally on the ribosome, a question arises as to whether the kinetics of translation influence the folding events in the growing nascent polypeptide chain. Translationally slow regions were identified on mRNAs for a set of 37 multidomain proteins from Escherichia coli with known three-dimensional structures. The frequencies of individual codons in mRNAs of highly expressed genes from E. coli were taken as a measure of codon translation speed. Analysis of codon usage in slow regions showed a consistency with the experimentally determined translation rates of codons; abundant codons that are translated with faster speeds compared with their synonymous codons were found to be avoided; rare codons that are translated at an unexpectedly higher rate were also found to be avoided in slow regions. The statistical significance of the occurrence of such slow regions on mRNA spans corresponding to the oligopeptide domain termini and linking regions on the encoded proteins was assessed. The amino acid type and the solvent accessibility of the residues coded by such slow regions were also examined. The results indicated that protein domain boundaries that mark higher-order structural organization are largely coded by translationally slow regions on the RNA and are composed of such amino acids that are stickier to the ribosome channel through which the synthesized polypeptide chain emerges into the cytoplasm. The translationally slow nucleotide regions on mRNA possess the potential to form hairpin secondary structures and such structures could further slow the movement of ribosome. The results point to an intriguing correlation between protein synthesis machinery and in vivo protein folding. Examination of available mutagenic data indicated that the effects of some of the reported mutations were consistent with our hypothesis.

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↗

PubMed1994

Analysis of a translational enhancer upstream from the coat protein open reading frame of potato virus S.

Evidence has suggested that the subgenomic RNA of the carlavirus potato virus S is an efficient message for the coat protein, even though evidence suggests it is uncapped at its 5' terminus. We have investigated the effect of the upstream region of the coat protein gene of potato virus S on the level of reporter gene expression in vitro. The region of 101 nucleotides upstream of the coat protein, designated VTE (viral translational enhancer) was found to increase levels of translation in comparison to a synthetic leader when linked to the beta-glucuronidase (GUS) reporter gene in vitro in rabbit reticulocyte and wheat germ lysate. VTE was also able to increase translation of the reporter gene luciferase (LUC) in vitro above the levels obtained for both a synthetic leader and a leader obtained from a plant gene isolated from Arabidopsis thaliana. The level of enhancement was evident with both capped and uncapped transcripts. When the VTE sequence was deleted to 20 nucleotides of the upstream region, thus removing the nucleotide block homologous among carlaviruses, the ability to enhance levels of translation was removed. In vitro translation studies indicated that the translational enhancement activity of VTE was at least partially cap independent. Translation of VTE linked to reporter genes in the presence of cap analogue was relatively unaffected whereas synthetic leader and a plant leader constructs were both more sensitive. In vitro competition analysis revealed that when short RNA transcripts representing the 101 nucleotides of VTE were added in trans to functional VTE leader LUC constructs there was a marked decrease in the level of translation when compared with a synthetic leader added in trans. These results suggest that the upstream region of the coat protein ORF of potato virus S promotes translation in a cap-independent manner that may involve the binding of proteins and/or ribosomes to the 101 nucleotides of the VTE sequence.

Animals↗