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PubMed1992

Synthesis of chlorophyll a regulates translation of chlorophyll a apoproteins P700, CP47, CP43 and D2 in barley etioplasts.

Accumulation of plastid-encoded chlorophyll apoproteins and chlorophyll synthesis are controlled by light in angiosperms. An in vitro system utilizing isolated and lysed barley (Hordeum vulgare L.) etioplasts revealed the specific accumulation of P700, CP47, CP43 and D2 triggered by de novo synthesis of chlorophyll. Accumulation rates of radiolabelled chlorophyll apoproteins were linear for about 30 min. Pulse/chase translation assays showed that synthesis of chlorophyll does not result in increased chlorophyll apoprotein stability. Instead turnover rates of chlorophyll apoproteins were higher in the presence than in the absence of chlorophyll. Chlorophyll-dependent accumulation of chlorophyll apoproteins must therefore be regulated on the level of translation. Translation of chlorophyll apoproteins was blocked to about 50% by addition of 30-50 microM aurintricarboxylic acid or 20 microM kasugamycin. The kinetics of chlorophyll-dependent translation indicated that the in vitro translation system is capable of translation initiation. The capability of translation initiation was lost in lysed etioplasts after preincubation for at least 5 min without chlorophyll synthesis. The results suggest that initiation is involved in chlorophyll-dependent regulation of translation.

Aminoglycosides↗

PubMed1997

Transcriptional and translational adjustments of psbA gene expression in mature chloroplasts during photoinhibition and subsequent repair of photosystem II.

The D1 reaction centre protein of photosystem II (PSII), encoded by the plastid psbA gene, has the highest turnover rate of all thylakoid proteins, due to light-induced damage to D1. The expression of the psbA gene was studied in chloroplasts of fully developed pea (Pisum sativum L.) leaves during high-light photoinhibitory treatment and subsequent restoration of PSII function at low light. psbA transcript levels were determined and the translational activity was followed by in vivo pulse-labelling, by in vitro translations with intact chloroplasts, and by run-off translations on isolated thylakoid membranes. PSII photochemical efficiency was determined in vivo by monitoring the ratio of variable fluorescence to maximal fluorescence (F(V)/F(M)). Enhanced D1 synthesis in pea leaves, upon a shift first from darkness to growth light and subsequently to high light, was accompanied by a substantial increase in the total number of pshA transcripts and by the accumulation of psbA mRNA x initiation complexes on thylakoid membrane. This suggested that high-light illumination increased the transcriptional activity of the psbA gene in mature leaves, and that enhanced translational initiation of psbA mRNA was followed by docking of the initiation complexes to the thylakoid membrane. The high-light-induced increase in the number of thylakoid-associated psbA mRNA x initiation complexes, occurred in parallel with enhanced in vivo D1 synthesis. This, however, did not result in an enhanced accumulation of D1 translation products in in vitro run-off translations when pea leaves were shifted from growth light to high light. This may suggest that at high light only a portion of thylakoid-associated psbA mRNA can be under translational elongation at a given moment. When the leaves were shifted from high light to low light to allow repair of PSII, thylakoid-associated psbA mRNA was rapidly released from the membrane and the high-light-induced pool of psbA transcripts was degraded. The synthesis of the D1 protein decreased on the same time scale. However, the restoration of PSII photochemical function, measured as F(V)/F(M), took a substantially longer time. It is concluded that during changing light conditions, mature leaves are able to adjust psbA gene expression both at the transcriptional and at the translational level.

Chloroplasts↗

PubMedDate not supplied

An in-line optical image translator with applications in x-ray videography.

Many applications in radiography require, or would benefit from, the ability to translate, i.e. move, an optical image in the detector plane. In this paper, we describe the design and characterization of a prism-based optical image translator for insertion into existing XRII-video imaging systems. A pair of prisms rotatable about the optical axis form a very compact in-line optical image translator for installation in the parallel light path between an x-ray image intensifier and its video camera. Rotation of the prisms translates the XRII optical image on the camera target. With the addition of x-ray and light collimators to limit the image to a single video line, x-ray streak images may be acquired. By rotating an object in the x-ray beam during a streak, a complete computed tomography (CT) data set may be acquired. This image translator can translate an image anywhere in the focal plane of a 50-mm-output lens within a 40-mm-diam circle. The prisms have an aperture of 50 mm, permitting an optical speed of F/2 with a 50-mm output lens. The design is insensitive to angular alignment errors. This image translator is achromatic, since the spectral width of the output phosphorus of image intensifiers is sufficient to introduce blurring in a nonacrhomatic design. A prism-based image translator introduces image distortion, since the prisms do not operate at minimum deviation. The distortion is less than 4% over all parts of a typical detector area, and less than 1% in the central region of the image.(ABSTRACT TRUNCATED AT 250 WORDS)

Biophysical Phenomena↗

PubMed2001

Inhibition of corticotropin releasing hormone type-1 receptor translation by an upstream AUG triplet in the 5' untranslated region.

The influence of an upstream open reading frame (ORF) in the 5'-untranslated region (UTR) of the mRNA on corticotropin-releasing hormone receptor type 1 (CRHR1) translation was studied in constructs containing the 5'-UTR of CRHR1, with or without an ATG-to-ATA mutation in the upstream ORF, and the main ORF of luciferase or CRHR1. Upstream mutation in luciferase constructs increased luciferase activity when transfected into COS-7 or AtT20 cells compared with the native 5'-UTR. Transfection of CRHR1 constructs containing the upstream mutation into AtT20 or LVIP2.0zc reporter cells, resulted in higher (125)I-Tyr-oCRH binding and corticotropin-releasing hormone-stimulated cAMP production, without changes in CRHR1 mRNA levels (measured by RNase protection assay). In vitro translation of luciferase or CRHR constructs with or without mutation of the upstream ATG, and Western blot analysis with anti-luciferase and anti-CRHR1 antibodies confirmed that mutation of the upstream ATG increases translation of the main ORF. The mechanism by which the upstream ORF inhibits translation may involve translation of the upstream peptide, because in vitro translation, or transfection into LVIP2.0zc cells of a fusion construct of the upstream ORF and green fluorescent protein (GFP) yielded a band consistent with the molecular size of GFP protein. The study shows that the upstream AUG in 5'-UTR of CRHR1 mRNA inhibits receptor expression by inhibiting mRNA translation and suggests the short open reading frame in the 5'-UTR plays a role in regulating translation of the CRH receptor.

5' Untranslated Regions↗

PubMed2006

A variation of the translation attenuation model can explain the inducible regulation of the pBC16 tetracycline resistance gene in Bacillus subtilis.

Expression of the tet resistance gene from plasmid pBC16 is induced by the antibiotic tetracycline, and induction is independent of the native promoter for the gene. The nucleotide sequence at the 5' end of the tet mRNA (the leader region) is predicted to assume a complex secondary structure that sequesters the ribosome binding site for the tet gene. A spontaneous, constitutively expressed tet gene variant contains a mutation predicted to provide the tet gene with a nonsequestered ribosome binding site. Lastly, comparable levels of tet mRNA can be demonstrated in tetracycline-induced and uninduced cells. These results are consistent with the idea that the pBC16 tet gene is regulated by translation attenuation, a model originally proposed to explain the inducible regulation of the cat and erm genes in gram-positive bacteria. As with inducible cat and erm genes, the pBC16 tet gene is preceded by a translated leader open reading frame consisting of a consensus ribosome binding site and an ATG initiation codon, followed by 19 sense codons and a stop codon. Mutations that block translation of cat and erm leaders prevent gene expression. In contrast, we show that mutations that block translation of the tet leader result in constitutive expression. We provide evidence that translation of the tet leader peptide coding region blocks tet expression by preventing the formation of a secondary-structure complex that would, in the absence of leader translation, expose the tet ribosome binding site. Tetracycline is proposed to induce tet by blocking or slowing leader translation. The results indicate that tet regulation is a variation of the translation attenuation model.

Amino Acid Sequence↗

PubMed1998

Translation of the flagellar gene fliO of Salmonella typhimurium from putative tandem starts.

The flagellar gene fliO of Salmonella typhimurium can be translated from an AUG codon that overlaps the termination codon of fliN (K. Ohnishi et al., J. Bacteriol. 179:6092-6099, 1997). However, it had been concluded on the basis of complementation analysis that in Escherichia coli a second start codon 60 bp downstream was the authentic one (J. Malakooti et al., J. Bacteriol. 176:189-197, 1994). This raised the possibility of tandem translational starts, such as occur for the chemotaxis gene cheA; this possibility was increased by the existence of a stem-loop sequence covering the second start, a feature also found with cheA. Protein translated from the first start codon was detected regardless of whether the second start codon was present; it was also detected when the stem-loop structure was disrupted or deleted. Translation from the second start codon, either as the natural one (GUG) or as AUG, was not detected when the first start and intervening sequence were intact. Nor was it detected when the first codon was attenuated (by conversion of AUGAUG to AUAAUA; in S. typhimurium there is a second, adjacent, AUG) or eliminated (by conversion to CGCCGC); disruption of the stem-loop structure still did not yield detectable translation from the second start. When the entire sequence up to the second start was deleted, translation from the second start was detected provided the natural codon GUG had been converted to AUG. A fliO null mutant could be fully complemented in swarm assays whenever the first start and intervening sequence were present, regardless of the state of the second start. Reasonably good complementation occurred when the first start and intervening sequence were absent provided the second start was intact, either as AUG or as GUG; thus translation from the GUG codon must have been occurring even though protein levels were too low to be detected. The translated intervening sequence is rather divergent between S. typhimurium and E. coli and corresponds to a substantial cytoplasmic domain prior to the sole transmembrane segment, which is highly conserved; the sequence following the second start begins immediately prior to that transmembrane segment. The significance of the data for FliO is discussed and compared to the equivalent data for CheA. Attention is also drawn to the fact that given an optimal ribosome binding site, AUA can serve as a fairly efficient start codon even though it seldom if ever appears to be used in nature.

Amino Acid Sequence↗

PubMed2002

Critical regions of secM that control its translation and secretion and promote secretion-specific secA regulation.

SecA is an essential ATP-driven motor protein that binds to presecretory or membrane proteins and the translocon and promotes the translocation or membrane integration of these proteins. secA is subject to a protein secretion-specific form of regulation, whereby its translation is elevated during secretion-limiting conditions. A novel mechanism that promotes this regulation involves translational pausing within the gene upstream of secA, secM. The secM translational pause prevents formation of an RNA helix that normally blocks secA translational initiation. The duration of this pause is controlled by the rate of secretion of nascent SecM, which in turn depends on its signal peptide and a functional translocon. We characterized the atypical secM signal peptide and found that mutations within the amino-terminal region specifically affect the secM translational pause and secA regulation, while mutations in the hydrophobic core region affect SecM secretion as well as translational pausing and secA regulation. In addition, mutational analysis of the 3' end of secM allowed us to identify a conserved region that is required to promote the translational pause that appears to be operative at the peptide level. Together, our results provide direct support for the secM translational pause model of secA regulation, and they pinpoint key sequences within secM that promote this important regulatory system.

3' Untranslated Regions↗

PubMed2004

The trp RNA-binding attenuation protein of Bacillus subtilis regulates translation of the tryptophan transport gene trpP (yhaG) by blocking ribosome binding.

Expression of the Bacillus subtilis tryptophan biosynthetic genes (trpEDCFBA and pabA [trpG]) is regulated in response to tryptophan by TRAP, the trp RNA-binding attenuation protein. TRAP-mediated regulation of the tryptophan biosynthetic genes includes a transcription attenuation and two distinct translation control mechanisms. TRAP also regulates translation of trpP (yhaG), a single-gene operon that encodes a putative tryptophan transporter. Its translation initiation region contains triplet repeats typical of TRAP-regulated mRNAs. We found that regulation of trpP and pabA is unaltered in a rho mutant strain. Results from filter binding and gel mobility shift assays demonstrated that TRAP binds specifically to a segment of the trpP transcript that includes the untranslated leader and translation initiation region. While the affinities of TRAP for the trpP and pabA transcripts are similar, TRAP-mediated translation control of trpP is much more extensive than for pabA. RNA footprinting revealed that the trpP TRAP binding site consists of nine triplet repeats (five GAG, three UAG, and one AAG) that surround and overlap the trpP Shine-Dalgarno (S-D) sequence and translation start codon. Results from toeprint and RNA-directed cell-free translation experiments indicated that tryptophan-activated TRAP inhibits TrpP synthesis by preventing binding of a 30S ribosomal subunit. Taken together, our results establish that TRAP regulates translation of trpP by blocking ribosome binding. Thus, TRAP coordinately regulates tryptophan synthesis and transport by three distinct mechanisms: attenuation transcription of the trpEDCFBA operon, promoting formation of the trpE S-D blocking hairpin, and blocking ribosome binding to the pabA and trpP transcripts.

Amino Acid Transport Systems↗

PubMed1980

Translational control of protein synthesis after infection by vesicular stomatitis virus.

Four hours after infection of BHK cells by vesicular stomatitis virus (VSV), the rate of total protein synthesis was about 65% that of uninfected cells and synthesis of the 12 to 15 predominant cellular polypeptides was reduced to a level about 25% that of control cells. As determined by in vitro translation of isolated RNA and both one- and two-dimensional gel analyses of the products, all predominant cellular mRNA's remained intact and translatable after infection. The total amount of translatable mRNA per cell increased about threefold after infection; this additional mRNA directed synthesis of the five VSV structural proteins. To determine the subcellular localization of cellular and viral mRNA before and after infection, RNA from various sizes of polysomes and nonpolysomal ribonucleoproteins (RNPs) was isolated from infected and noninfected cells and translated in vitro. Over 80% of most predominant species of cellular mRNA was bound to polysomes in control cells, and over 60% was bound in infected cells. Only 2 of the 12 predominant species of translatable cellular mRNA's were localized to the RNP fraction, both in infected and in uninfected cells. The average size of polysomes translating individual cellular mRNA's was reduced about two- to threefold after infection. For example, in uninfected cells, actin (molecular weight 42,000) mRNA was found predominantly on polysomes with 12 ribosomes; after infection it was found on polysomes with five ribosomes, the same size of polysomes that were translating VSV N (molecular weight 52,000) and M (molecular weight 35,000) mRNA. We conclude that the inhibition of cellular protein synthesis after VSV infection is due, in large measure, to competition for ribosomes by a large excess of viral mRNA. The efficiency of initiation of translation on cellular and viral mRNA's is about the same in infected cells; cellular ribosomes are simply distributed among more mRNA's than are present in growing cells. About 20 to 30% of each of the predominant cellular and viral mRNA's were present in RNP particles in infected cells and were presumably inactive in protein synthesis. There was no preferential sequestration of cellular or viral mRNA's in RNPs after infection.

Animals↗

PubMed1987

Restriction of translation of capped mRNA in vitro as a model for poliovirus-induced inhibition of host cell protein synthesis: relationship to p220 cleavage.

Poliovirus infection of HeLa cells results in a rapid inhibition of host protein synthesis by a mechanism that does not affect the translation of poliovirus RNA. It has been suggested that this virus-induced translational control results from inactivation of the cap-binding protein complex, and it has been shown that the 220-kilodalton component(s) (p220) of the cap-binding protein complex is cleaved in infected HeLa cells to form antigenically related polypeptides of 100 to 130 kilodaltons. We have previously described an activity in infected cells that specifically restricts translation of capped mRNA in rabbit reticulocyte lysates. Here, we describe further refinements and characterization of restriction assay. We determined that the assay is a good in vitro model for study of host cell shutoff by several criteria: (i) translation was inhibited in both instances at the step involving mRNA binding to ribosomes; (ii) translation of capped mRNA was specifically inhibited, whereas translation of poliovirus RNA was not; (iii) restriction activity appeared in infected cells with kinetics which parallel host cell shutoff; and (iv) restriction activity, like the specific inhibition of host translation, appeared in cells infected in the presence of guanidine-HCl. The restricting activity was partially purified from poliovirus-infected cells and was compared with the virus-induced p220 cleavage activity. Both activities copurified through numerous cell fractionation and biochemical fractionation procedures. However, specific restriction of capped mRNA translation in reticulocyte lysates occurred without complete cleavage of the endogenous p220.

Animals↗

PubMed1994

La autoantigen alleviates translational repression by the 5' leader sequence of the human immunodeficiency virus type 1 mRNA.

The trans-activation response element (TAR) at the 5' end of the human immunodeficiency virus type 1 (HIV-1) mRNAs forms a stable hairpin structure which is a target for binding of the virally encoded protein Tat, which activates viral gene expression, as well as several cellular factors. TAR is also inhibitory to translation. One of several host factors that binds to TAR RNA is the La autoantigen, an RNA-binding protein which functions in RNA polymerase III transcription termination and has also been implicated in cap-independent internal translation initiation on poliovirus RNA. Here we show that La autoantigen alleviates translational repression by the HIV-1 leader RNA. In rabbit reticulocyte lysate, La relieves the cis-inhibitory effect of the TAR RNA on translation of bacterial chloramphenicol acetyltransferase (CAT) mRNA but not inhibition that is mediated by an artificial secondary structure element. Canonical translation factors exhibited slight (eIF-2 and GEF) or no (eIF-4A, eIF-4B, eIF-4E, eIF-4F, eIF-3, and eEF-1 alpha) stimulatory activity on translation of TAR-containing CAT mRNA. In addition, we show that poliovirus RNA, in spite of being an inefficient template in rabbit reticulocyte lysate, is a strong competitive inhibitor of translation of TAR-containing CAT mRNA but not CAT mRNA. This inhibition can be relieved by La but not by any other translation factor. The results suggest a possible involvement of the La autoantigen in HIV-1 gene expression.

Autoantigens↗

PubMed1995

Translation of the human papillomavirus type 16 E7 oncoprotein from bicistronic mRNA is independent of splicing events within the E6 open reading frame.

In this study we investigated the translational capacities of bicistronic and spliced mRNAs originating from the E6 and E7 regions of the high-risk genital human papillomavirus type 16 (HPV-16) and the low-risk HPV-11. For HPV-16 it was found, unexpectedly, that E7 protein could be translated from full-length bicistronic E6-E7 mRNAs. E6*I and E6*II splicing events were not required for E7 synthesis, nor did splicing increase the efficiency of E7 translation significantly. In cells, E7 synthesis from all known naturally occurring mRNA structures was very inefficient compared with that from synthetic monocistronic controls, suggesting that HPV-16 employs translational mechanisms to restrict E7 protein levels. For HPV-11, only RNAs initiated at the P264 promoter, located within the E6 open reading frame, were capable of providing an efficient template for E7 synthesis. P264-initiated mRNAs were as efficient in vivo as monocistronic controls, suggesting that the low-risk HPV-11 does not limit E7 synthesis by translational mechanisms. A detailed analysis of HPV-16 templates by using site-directed mutagenesis showed that the majority of ribosomes which ultimately translate E7 have not reinitiated after translating some or all of the upstream open reading frames. The data support a model in which the failure of 40S ribosomal initiation complexes to recognize the E6 AUG renders them capable of proceeding efficiently to translate E7.

Animals↗

PubMed1995

Translation but not the encoded sequence is essential for the efficient propagation of the defective interfering RNAs of the coronavirus mouse hepatitis virus.

The defective interfering (DI) RNA MIDI of mouse hepatitis virus strain A59 (MHV-A59) contains a large open reading frame (ORF) spanning almost its entire genome. This ORF consists of sequences derived from ORF1a, ORF1b, and the nucleocapsid gene. We have previously demonstrated that mutations that disrupt the ORF decrease the fitness of MIDI and its derivatives (R. J. de Groot, R. G. van der Most, and W. J. M. Spaan, J. Virol. 66:5898-5905, 1992). To determine whether translation of the ORF per se is required or whether the encoded polypeptide or a specific sequence is involved, we analyzed sets of related DI RNAs containing different ORFs. After partial deletion of ORF1b and nucleocapsid gene sequences, disruption of the remaining ORF is still lethal; translation of the entire ORF is not essential, however. When a large fragment of the MHV-A59 spike gene, which is not present in any of the MHV-A59 DI RNAs identified so far, was inserted in-frame into a MIDI derivative, translation across this sequence was vital to DI RNA survival. Thus, the translated sequence is irrelevant, indicating that translation per se plays a crucial role in DI virus propagation. Next, it was examined during which step of the viral life cycle translation plays its role. Since the requirement for translation also exists in DI RNA-transfected and MHV-infected cells, it follows that either the synthesis or degradation of DI RNAs is affected by translation.

Base Sequence↗

PubMed1995

Efficiency of reinitiation of translation on human immunodeficiency virus type 1 mRNAs is determined by the length of the upstream open reading frame and by intercistronic distance.

In this study, we examined the mechanism of translation of the human immunodeficiency virus type 1 tat mRNA in eucaryotic cells. This mRNA contains the tat open reading frame (ORF), followed by rev and nef ORFs, but only the first ORF, encoding tat, is efficiently translated. Introduction of premature stop codons in the tat ORF resulted in efficient translation of the downstream rev ORF. We show that the degree of inhibition of translation of rev is proportional to the length of the upstream tat ORF. An upstream ORF spanning 84 nucleotides was predicted to inhibit 50% of the ribosomes from initiating translation at downstream AUGs. Interestingly, the distance between the upstream ORF and the start codon of the second ORF also played a role in efficiency of downstream translation initiation. It remains to be investigated if these conclusions relate to translation of mRNAs other than human immunodeficiency virus type 1 mRNAs. The strong inhibition of rev translation exerted by the presence of the tat ORF may reflect the different roles of Tat and Rev in the viral life cycle. Tat acts early to induce high production of all viral mRNAs. Rev induces a switch from the early to the late phase of the viral life cycle, resulting in production of viral structural proteins and virions. Premature Rev production may result in entrance into the late phase in the presence of suboptimal levels of viral mRNAs coding for structural proteins, resulting in inefficient virus production.

Base Sequence↗

PubMed1998

Role of Rta in the translation of bicistronic BZLF1 of Epstein-Barr virus.

The BZLF1 gene of Epstein-Barr virus (EBV), which encodes a transcription factor, Zta, is transcribed into monocistronic and bicistronic mRNAs from two different promoters during the immediate-early stage of the EBV lytic cycle. It is generally accepted that the Zta protein translated from the monocistronic mRNA profoundly influences the activation of the EBV lytic cycle. In this study, we constructed a plasmid, pCMV-RZLUC, which can transcribe a bicistronic mRNA consisting of BRLF1 and a BZLF1-luc fusion gene under latent conditions. P3HR1 cells transfected with this plasmid produce a luciferase activity which is approximately 17-fold higher than the activity exhibited by pRZLUC, a plasmid incapable of transcribing the bicistronic mRNA. Genetic analyses indicated that mutations in BRLF1 not only can decrease the translation of the fusion gene from the bicistronic mRNA but can also be complemented by a functional BRLF1 gene in cis. This observation implies that the product of BRLF1, Rta, is involved in the translation of the downstream gene. Results presented herein also demonstrate that these mutations cannot be complemented in trans with a plasmid overexpressing Rta, suggesting that the amount of Rta in the vicinity of the intercistronic region may be crucial for the translation. Furthermore, our results correspond to those of previous investigations indicating that the Zta protein can be translated from the bicistronic mRNA and that, similar to the translation of bicistronic ZLUC, mutations in BRLF1 also hinder the translation of Zta from the BRLF1-BZLF1 bicistronic mRNA. Translation of Zta from the bicistronic mRNA may play an essential role in the activation of the EBV lytic cycle.

Animals↗

PubMed1999

Natural variation in translational activities of the 5' nontranslated RNAs of hepatitis C virus genotypes 1a and 1b: evidence for a long-range RNA-RNA interaction outside of the internal ribosomal entry site.

The 5' nontranslated RNA (5'NTR) of a genotype 1b hepatitis C virus (HCV-N) directs cap-independent translation of the HCV-N polyprotein with about twofold less efficiency than the 5'NTR of a genotype 1a virus under physiologic conditions (Hutchinson strain, or HCV-H) (M. Honda et al., Virology 222:31-42, 1996). Here, we show by mutational analysis that substitution of the AG dinucleotide sequence at nucleotides (nt) 34 and 35 of HCV-N with GA (present in HCV-H) restores the translational activity to that of the HCV-H 5'NTR both in vitro and in vivo. These nucleotides are located upstream of the minimal essential internal ribosome entry site (IRES), as a 6-nt deletion spanning nt 32 to 37 also increased the translational activity of the HCV-N 5'NTR to that of HCV-H. Thus, the upstream AG dinucleotide sequence has an inhibitory effect on IRES-directed translation. Surprisingly, however, this inhibitory effect was observed only when the translated, downstream RNA sequence contained nt 408 to 929 of HCV (capsid-coding RNA). Further analysis of RNA transcripts containing frameshift mutations demonstrated that the nucleotide sequence of the transcript, and not the amino acid sequence of the expressed capsid protein, determines this difference in translation efficiency. The difference between the translational activities of the HCV-N and HCV-H transcripts was increased when translation was carried out in reticulocyte lysates containing high K+ concentrations, with a sevenfold difference evident at 130 to 150 mM K+. These results suggest that there is an RNA-RNA interaction involving 5'NTR and capsid-coding sequences flanking the IRES and that this is responsible for the reduced IRES activity of the genotype 1b virus, HCV-N.

5' Untranslated Regions↗

PubMed2003

Cap-independent translational enhancement by the 3' untranslated region of red clover necrotic mosaic virus RNA1.

Red clover necrotic mosaic virus (RCNMV) is a member of the genus Dianthovirus and has a bipartite positive-sense genomic RNA with 3' ends that are not polyadenylated. In this study, we show that both genomic RNA1 and RNA2 lack a 5' cap structure and that uncapped in vitro transcripts of RCNMV RNA1 replicated to a level comparable to that for capped transcripts in cowpea protoplasts. Because the 5' cap and 3' poly(A) tail play important roles in the translation of many eukaryotic mRNAs, genomic RNAs of RCNMV should contain an element(s) responsible for 5' cap- and poly(A) tail-independent translation of viral protein. By using a luciferase reporter assay system in vivo, we showed that the 3' untranslated region (UTR) of RNA1 alone significantly enhanced translation of the luciferase reporter gene in the absence of the 5' cap structure. Deletion studies revealed that the middle region (between nucleotides 3596 and 3732) in the 3' UTR, designated the 3' translation element of Dianthovirus RNA1 (3'TE-DR1), plays an important role in cap-independent translation. This region contained a stem-loop structure conserved among members of the genera Dianthovirus and LUTEOVIRUS: A five-base substitution in the loop abolished cap-independent translational activity, as reported for a luteovirus, indicating that this stem-loop is one of the functional structures in the 3'TE-DR1 involved in cap-independent translation. Finally, we suggest that cap-independent translational activity is required for RCNMV RNA1 replication in protoplasts.

3' Untranslated Regions↗

PubMed2006

Cap-independent translation mechanism of red clover necrotic mosaic virus RNA2 differs from that of RNA1 and is linked to RNA replication.

The genome of Red clover necrotic mosaic virus (RCNMV) in the genus Dianthovirus is divided into two RNA molecules of RNA1 and RNA2, which have no cap structure at the 5' end and no poly(A) tail at the 3' end. The 3' untranslated region (3' UTR) of RCNMV RNA1 contains an essential RNA element (3'TE-DR1), which is required for cap-independent translation. In this study, we investigated a cap-independent translational mechanism of RNA2 using a firefly luciferase (Luc) gene expression assay system in cowpea protoplasts and a cell-free lysate (BYL) prepared from evacuolated tobacco BY2 protoplasts. We were unable to detect cis-acting RNA sequences in RNA2 that can replace the function of a cap structure, such as the 3'TE-DR1 of RNA1. However, the uncapped reporter RNA2, RNA2-Luc, in which the Luc open reading frame (ORF) was inserted between the 5' UTR and the movement protein ORF, was effectively translated in the presence of p27 and p88 in protoplasts in which RNA2-Luc was replicated. Time course experiments in protoplasts showed that the translational activity of RNA2-Luc did not reflect the amount of RNA2. Mutations in cis-acting RNA replication elements of RNA2 abolished the cap-independent translational activity of RNA2-Luc, suggesting that the translational activity of RNA2-Luc is coupled to RNA replication. Our results show that the translational mechanism differs between two segmented genomic RNAs of RCNMV. We present a model in which only RNA2 that is generated de novo through the viral RNA replication machinery functions as mRNA for translation.

Base Sequence↗