Eukaryotic elongation factor 1A interacts with the upstream pseudoknot domain in the 3' untranslated region of tobacco mosaic virus RNA

The genomic RNA of tobacco mosaic virus (TMV), like that of other positive-strand RNA viruses, acts as a template for both translation and replication. The highly structured 3' untranslated region (UTR) of TMV RNAs plays an important role in both processes; it is not polyadenylated but ends with a tRNA-like structure (TLS) preceded by a conserved upstream pseudoknot domain (UPD). The TLS of tobamoviral RNAs can be specifically aminoacylated and, in this state, can interact with eukaryotic elongation factor 1A (eEF1A)/GTP with high affinity. Using a UV cross-linking assay, we detected another specific binding site for eEF1A/GTP, within the UPDs of TMV and crucifer-infecting tobamovirus (crTMV), that does not require aminoacylation. A mutational analysis revealed that UPD pseudoknot conformation and some conserved primary sequence elements are required for this interaction. Its possible role in the regulation of tobamovirus gene expression and replication is discussed.     

Recognition of the tRNA-like structure in tobacco mosaic viral RNA by ATP/CTP:tRNA nucleotidyltransferases from Escherichia coli and Saccharomyces cerevisiae

The 3'-terminal tRNA-like structure of the tobacco mosaic virus RNA interacts with ATP/CTP:tRNA nucleotidyltransferases from Escherichia coli or yeast in much the same manner as do tRNAs. Primary sites of interaction cluster near the 3' end and in the loop proposed to be analogous to the psi-loop of a tRNA. Some modified bases in the tRNA-like structure inhibit interaction with nucleotidyltransferase, yet the analogous bases in a tRNA do not. The location of some of these nucleotides within the analog to the psi-loop suggests that this structure differs slightly from its counterpart in a tRNA. The location of other such bases in the helical stem near the 3' end can be explained if the pseudoknot is disrupted by these modified bases or if the tertiary structure of the RNA is altered in the enzyme-RNA complex. A partially denatured secondary structure that persists on denaturing gels is proposed.     

A phylogenetically conserved sequence within viral 3'' untranslated RNA pseudoknots regulates translation.

Both the 68-base 5' leader (omega) and the 205-base 3' untranslated region (UTR) of tobacco mosaic virus (TMV) promote efficient translation. A 35-base region within omega is necessary and sufficient for the regulation. Within the 3' UTR, a 52-base region, composed of two RNA pseudoknots, is required for regulation. These pseudoknots are phylogenetically conserved among seven viruses from two different viral groups and one satellite virus. The pseudoknots contained significant conservation at the secondary and tertiary levels and at several positions at the primary sequence level. Mutational analysis of the sequences determined that the primary sequence in several conserved positions, particularly within the third pseudoknot, was essential for function. The higher-order structure of the pseudoknots was also required. Both the leader and the pseudoknot region were specifically recognized by, and competed for, the same proteins in extracts made from carrot cell suspension cells and wheat germ. Binding of the proteins is much stronger to omega than the pseudoknot region. Synergism was observed between the TMV 3' UTR and the cap and to a lesser extent between omega and the 3' UTR. The functional synergism and the protein binding data suggest that the cap, TMV 5' leader, and 3' UTR interact to establish an efficient level of translation.     

The three-dimensional folding of the tRNA-like structure of tobacco mosaic virus RNA. A new building principle applied twice

The structure of the tRNA-like 3' terminus of tobacco mosaic virus (TMV) RNA has been studied. A 3' -terminal fragment possessing the tRNA-like properties was probed with chemical modification and enzymatic digestions. A model of the secondary structure is proposed for the last 105 nucleotides. The corresponding region of other tobamoviral RNAs can be folded in an identical secondary structure. A three-dimensional model for the tRNA-like structure is given which is compared with those proposed earlier for the tRNA-like 3' termini of turnip yellow mosaic virus (TYMV) RNA and brome mosaic virus (BMV) RNA. A new building principle which we discovered previously by studying the latter RNAs appears to be applied twice in the tRNA-like structure of TMV RNA. The determination of the minimal length requirement for recognition of CTP, ATP:tRNA nucleotidyl-transferase reveals a size of ˜100 nucleotides in agreement with the models proposed.     

Second-site suppressor mutations assist in studying the function of the 3'' noncoding region of turnip yellow mosaic virus RNA.

The 3' noncoding region of turnip yellow mosaic virus RNA includes an 82-nucleotide-long tRNA-like structure domain and a short upstream region that includes a potential pseudoknot overlapping the coat protein termination codon. Genomic RNAs with point mutations in the 3' noncoding region that result in poor replication in protoplasts and no systemic symptoms in planta were inoculated onto Chinese cabbage plants in an effort to obtain second-site suppressor mutations. Putative second-site suppressor mutations were identified by RNase protection and sequencing and were then introduced into genomic cDNA clones to permit their characterization. A C-57----U mutation in the tRNA-like structure was a strong suppressor of the C-55----A mutation which prevented both systemic infection and in vitro valylation of the viral RNA. Both of these phenotypes were rescued in the double mutant. An A-107----C mutation was a strong second-site suppressor of the U-96----G mutation, permitting the double mutant to establish systemic infection. The C-107 and G-96 mutations are located on opposite strands of one helix of a potential pseudoknot, and the results support a functional role for the pseudoknot structure. A mutation near the 5' end of the genome (G + 92----A), at position -3 relative to the initiation codon of the essential open reading frame 206, was found to be a general potentiator of viral replication, probably as a result of enhanced expression of open reading frame 206. The A + 92 mutation enhanced the replication of mutant TYMC-G96 in protoplasts but was not a sufficiently potent suppressor to permit systemic spread of the A + 92/G-96 double mutant in plants.     

The turnip yellow mosaic virus tRNA-like structure cannot be replaced by generic tRNA-like elements or by heterologous 3' untranslated regions known to enhance mRNA expression and stability.

The tRNA-like structure (TLS) at the 3' end of the turnip yellow mosaic virus genome was replaced with heterologous tRNA-like elements, and with a poly(A) tail, in order to assess its role. Replacement with the valylatable TLSs from two closely related tymoviruses resulted in infectious viruses. In contrast, no systemic symptoms on plants, and only low viral accumulations in protoplasts, were observed for three chimeric genomes with 3' sequences known to enhance mRNA stability and translatability. One of these chimeras had a poly(A) tail, and the others had the TLS with associated upstream pseudoknot tracts from the 3' ends of brome mosaic and tobacco mosaic viruses. The latter two chimeric RNAs were shown to be appropriately folded by demonstrating their aminoacylation in vitro with tyrosine and histidine, respectively. The results show that enhancement of genome stability or gene expression is not the major role of the turnip yellow mosaic virus TLS. The major role is likely to be replicational, dependent on features present in tymoviral TLSs but not in generic tRNA-like structures.     

Functional and structural analysis of a pseudoknot upstream of the tag-encoded sequence in E. coli tmRNA

Escherichia coli tmRNA (transfer-messenger RNA) facilitates a trans-translation reaction in which a stalled ribosome on a terminatorless mRNA switches to an internal coding sequence in tmRNA, resulting in the addition of an 11 amino acid residue tag to the truncated protein that is a signal for degradation and in recycling of the stalled ribosome. A tmRNA secondary structure model with a partial tRNA-like structure and several pseudoknots was recently proposed. This report describes an extensive mutational analysis of one predicted pseudoknot (PK1) located upstream of the E. coli tmRNA tag-encoded sequence. Both the extent of aminoacylation and the alanine incorporation into the tag sequence, reflecting the two functions of tmRNA, were measured in vitro for all the engineered RNA variants. To characterize structure-function relationships for the tmRNA mutants, their solution conformations were investigated by using structural probes and by measuring the temperature dependence of their UV absorbance. This analysis strongly supports the presence of a pseudoknot in E. coli tmRNA, and its involvement in trans-translation. Mutations disrupting the first stem of the pseudoknot inactivate function and promote stable alternative conformations. Mutations of the second stem of the pseudoknot also effect both functions. The nucleotide stretch between the two stems (loop 2) is required for efficient trans-translation, and nucleotides at positions 61 and 62 must be guanine residues. The probing data suggest the presence of magnesium ion(s) interacting with loop 2. The loops crossing the minor and major grooves can be mutated without significant effects on tmRNA function. Nucleotide insertion or deletion between the pseudoknot and the coding sequence do not change the mRNA frame of the tag-peptide sequence, suggesting that the pseudoknot structure is not a determinant for the resumption of translation.     

An NMR and mutational analysis of an RNA pseudoknot of Escherichia coli tmRNA involved in trans-translation.

Transfer-messenger RNA (tmRNA) is a unique molecule that combines properties from both tRNA and mRNA, and facilitates a novel translation reaction termed trans -translation. According to phylogenetic sequence analysis among various bacteria and chemical probing analysis, the secondary structure of the 350-400 nt RNA is commonly characterized by a tRNA-like structure, and four pseudoknots with different sizes. A mutational analysis using a number of Escherichia coli tmRNA variants as well as a chemical probing analysis has recently demonstrated not only the presence of the smallest pseudoknot, PK1, upstream of the internal coding region, but also its direct implication in trans -translation. Here, NMR methods were used to investigate the structure of the 31 nt pseudoknot PK1 and its 11 mutants in which nucleotide substitutions are introduced into each of two stems or the linking loops. NMR results provide evidence that the PK1 RNA is folded into a pseudoknot structure in the presence of Mg(2+). Imino proton resonances were observed consistent with formation of two helical stem regions and these stems stacked to each other as often seen in pseudoknot structures, in spite of the existence of three intervening nucleo-tides, loop 3, between the stems. Structural instability of the pseudoknot structure, even in the presence of Mg(2+), was found in the PK1 mutants except in the loop 3 mutants which still maintained the pseudoknot folding. These results together with their biological activities indicate that trans -translation requires the pseudoknot structure stabilized by Mg(2+)and specific residues G61 and G62 in loop 3.     

Cap-independent translation of tobacco etch virus is conferred by an RNA pseudoknot in the 5'-leader

The tobacco etch virus (TEV) 5'-leader promotes cap-independent translation in a 5'-proximal position and promotes internal initiation when present in the intercistronic region of a dicistronic mRNA, indicating that the leader contains an internal ribosome entry site. The TEV 143-nucleotide 5'-leader folds into a structure that contains two domains, each of which contains an RNA pseudoknot. Mutational analysis of the TEV 5'-leader identified pseudoknot (PK) 1 within the 5'-proximal domain and an upstream single-stranded region flanking PK1 as necessary to promote cap-independent translation. Mutations to either stem or to loops 2 or 3 of PK1 substantially disrupted cap-independent translation. The sequence of loop 3 in PK1 is complementary to a region in 18 S rRNA that is conserved throughout eukaryotes. Mutations within L3 that disrupted its potential base pairing with 18 S rRNA reduced cap-independent translation, whereas mutations that maintained the potential for base pairing with 18 S rRNA had little effect. These results indicated that the TEV 5'-leader functionally substitutes for a 5'-cap and promotes cap-independent translation through a 45-nucleotide pseudoknot-containing domain.     

Temperature dependent chemical and enzymatic probing of the tRNA-like structure of TYMV RNA

In this paper we report on the thermal unfolding of the tRNA-like structure present at the 3' end of turnip yellow mosaic virus (TYMV) RNA. Diethyl pyrocarbonate (DEP), sodium bisulphite, nuclease S1 and ribonuclease T1 were used as structure probes at a broad range of temperatures. In this way most of the nucleotides present in the tRNA-like moiety were analysed. The melting behaviour of both secondary and tertiary interactions could be followed on the basis of the temperature dependent accessibility of the individual nucleotides or bases towards the various probes. The three-dimensional model of the tRNA-like domain (Dumas et al., J. Biomol. Struct. and Dyn. 4, 707 (1987] was supported by the results to a large extent. The interactions occurring between the T- and D-loop appear to be more complex than proposed in the latter model. Additional evidence for the presence of the RNA pseudoknot (Rietveld et al., Nucleic Acids Res. 10, 1929 (1982] was derived from the fact that the three coaxially stacked helical segments in the aminoacylacceptor arm displayed different melting transitions under certain experimental conditions. Aspects of melting behaviour and thermal stability of double helical regions within the tRNA-like structure are discussed, as well as the applicability of nucleases and modifying reagents at various temperatures in the analysis of RNA structure.     

Phylogenetic analysis of tmRNA secondary structure.

The bacterial tmRNA acts with dual tRNA-like and mRNA-like character to tag incomplete translation products for degradation. Comparative analysis of 17 tmRNA genes (including eight new sequences) has allowed us to deduce conserved features of the tmRNA secondary structure. Except in a segment that includes the first codon of the tag reading frame, tmRNA is highly structured, with four pseudoknots and a total of 11 conserved base pairing regions. The previously identified tRNA minihelix structure is connected by a long base paired region to a large structured domain composed of a pseudoknot, followed by the tag reading frame and a string of three rather similar pseudoknots. The conservation of numerous structural elements among diverse eubacterial species indicates that these elements have important function beyond simply forming an endonuclease-resistant link between the reading frame and the tRNA-like domain.     

Post-transcriptional regulation in higher eukaryotes: the role of the reporter gene in controlling expression

We have investigated whether reporter genes influence cytoplasmic regulation of gene expression in tobacco and Chinese hamster ovary (CHO) cells. Two genes, uidA encoding beta-glucuronidase (GUS) from Escherichia coli and Luc, encoding firefly luciferase (LUC), were used to analyze the ability of a cap, polyadenylated tail, and the 5'- and 3'-untranslated regions (UTR) from tobacco mosaic virus (TMV) to regulate expression. The regulation associated with the 5' cap structure and the TMV 5'-UTR, both of which enhance translational efficiency, was reporter gene-independent. The poly(A) tail and the TMV 3'-UTR, which is functionally equivalent to a poly(A) tail, increase translational efficiency as well as mRNA stability. The regulation associated with these 3' ends was highly reporter gene-dependent; their effect on GUS expression was almost an order of magnitude greater than that on LUC expression. In tobacco, the tenfold reporter gene effect on poly(A) tail or TMV 3'-UTR function could not be explained by a differential impact on mRNA stability; GUS and LUC mRNA half-life increased only twofold when either the poly(A) tail or TMV 3'-UTR was present. In CHO cells, however, GUS mRNA was stabilized to a greater extent by a poly(A) tail or the TMV 3'-UTR than was LUC mRNA.     

Insights into Factorless Translational Initiation by the tRNA-Like Pseudoknot Domain of a Viral IRES

The intergenic region internal ribosome entry site (IGR IRES) of the Dicistroviridae family adopts an overlapping triple pseudoknot structure to directly recruit the 80S ribosome in the absence of initiation factors. The pseudoknot I (PKI) domain of the IRES mimics a tRNA-like codon:anticodon interaction in the ribosomal P site to direct translation initiation from a non-AUG initiation codon in the A site. In this study, we have performed a comprehensive mutational analysis of this region to delineate the molecular parameters that drive IRES translation. We demonstrate that IRES-mediated translation can initiate at an alternate adjacent and overlapping start site, provided that basepairing interactions within PKI remain intact. Consistent with this, IGR IRES translation tolerates increases in the variable loop region that connects the anticodon- and codon-like elements within the PKI domain, as IRES activity remains relatively robust up to a 4-nucleotide insertion in this region. Finally, elements from an authentic tRNA anticodon stem-loop can functionally supplant corresponding regions within PKI. These results verify the importance of the codon:anticodon interaction of the PKI domain and further define the specific elements within the tRNA-like domain that contribute to optimal initiator Met-tRNA_i-independent IRES translation.     

Idiosyncratic behaviour of tRNA-like structures in translation of plant viral RNA genomes

Tobacco mosaic virus (TMV) and Nemesia ring necrosis virus (NeRNV) belong to the Tobamoviridae and Tymoviridae families, respectively. Although their RNAs present different 5'-untranslated regions and different family-specific genomic organizations, they share common 3'-ends organized into three consecutive pseudoknot structures followed by a histidylatable tRNA-like structure (TLS). We investigate here whether the histidine residue becomes incorporated into viral proteins and if the TLSs of TMV and NeRNV play a role in viral translation. Our results indicate that, regardless of the genomic context, the histidine moiety does not become incorporated in proteins via ribosomal translation, and that disruption of the TLS in either viral RNA does not perturb the viral translation patterns. In the light of the present data and of previous results on tymoviral TLSVal and bromoviral TLSTyr showing differential effects on translation, we suggest that the key role for the TLS in promoting translation initiation appears to be dictated by the TLS architecture and identity.     

Mutational analysis of the tRNA mimicry of brome mosaic virus RNA. Sequence and structural requirements for aminoacylation and 3'-adenylation

The genomic RNAs of brome mosaic virus (BMV) exhibit various tRNA-like properties, including specific tyrosylation by tyrosyl-tRNA synthetases and adenylation of the 3'-CCOH derivative by tRNA nucleotidyl transferases. We have studied the effect of numerous mutations in all domains of the tRNA-like structure of BMV RNA on tyrosylation and adenylation in vitro. Surprisingly few mutations resulted in more than 50% decrease in tyrosylation rates with either wheat germ or yeast synthetases; those mutations were at the 3' terminus, the pseudoknot, and the bases of arms B and E. The results suggest an interaction of synthetase with arm A as the analog of the aminoacyl acceptor stem of tRNAs, and arm B as the analog of the anticodon arm of tRNAs, although there is no apparent interaction with the terminal loop of arm B analogous to the interaction with the anticodon in tRNAs. Mutations at several loci resulted in large losses of adenylation activity catalyzed by wheat germ and Escherichia coli nucleotidyl transferases; those loci were the pseudoknot, the bases of arms B, C and D, and at the junctions of these arms with arm A. These studies have identified mutants specifically defective in one of the tRNA-like activities, which are appropriate for investigating the role of these activities during infection in vivo.     

Multi-domain Packing in the Aminoacylatable 3′ End of a Plant Viral RNA

Turnip yellow mosaic virus (TYMV) contains a tRNA-like structure (TLS) in its 3′ untranslated region (3′ UTR).  This highly structured element induces valylation of the viral RNA by host cell enzymes and is important for virus proliferation. Directly upstream of the TYMV TLS is an upstream pseudoknot domain (UPD) that has been considered to be structurally distinct from the TLS.  However, using a combination of functional, biochemical, and biophysical assays, we show that the entire 3′ UTR of the viral genome is a single structured element in the absence of cellular protein.  This packing architecture stabilizes the RNA structure and creates a better substrate for aminoacylation, and thus the UPD and TLS are functionally and structurally coupled.  It has been proposed that the TYMV TLS acts as a molecular switch between translation and replication. Our results suggest that this putative switch could be based on structural changes within the global architecture of the UTR induced by interactions with the ribosome. The TYMV TLS·UPD might demonstrate how RNA structural plasticity can play a role in regulation of biological processes.     

Selection of viral RNA-derived tRNA-like structures with improved valylation activities

The tRNA-like structure (TLS) of turnip yellow mosaic virus (TYMV) RNA was previously shown to be efficiently charged by yeast valyl-tRNA synthetase (ValRS). This RNA has a noncanonical structure at its 3'-terminus but mimics a tRNA L-shaped fold, including an anticodon loop containing the major identity nucleotides for valylation, and a pseudoknotted amino acid accepting domain. Here we describe an in vitro selection experiment aimed (i) to verify the completeness of the valine identity set, (ii) to elucidate the impact of the pseudoknot on valylation, and (iii) to investigate whether functional communication exists between the two distal anticodon and amino acid accepting domains. Valylatable variants were selected from a pool of 2 x 10(13) RNA molecules derived from the TYMV TLS randomized in the anticodon loop nucleotides and in the length (1-6 nucleotides) and sequence of the pseudoknot loop L1. After nine rounds of selection by aminoacylation, 42 have been isolated. Among them, 17 RNAs could be efficiently charged by yeast ValRS. Their sequence revealed strong conservation of the second and the third anticodon triplet positions (A(56), C(55)) and the very 3'-end loop nucleotide C(53). A large variability of the other nucleotides of the loop was observed and no wild-type sequence was recovered. The selected molecules presented pseudoknot domains with loop L1 varying in size from 3-6 nucleotides and some sequence conservation, but did neither reveal the wild-type combination. All selected variants are 5-50 times more efficiently valylated than the wild-type TLS, suggesting that the natural viral sequence has emerged from a combination of evolutionary pressures among which aminoacylation was not predominant. This is in line with the role of the TLS in viral replication.     

Cooperative thermal denaturation of the assembly origin region of TMV RNA

The assembly origin (AO) region of the tobacco mosaic virus RNA melts in an usually narrow (2.5 degrees C) temperature range. In an 0.01 M phosphate buffer the melting temperature of AO was found to be 41.5 degrees C. This value corresponds to the regions with the most stable secondary/tertiary structure of the whole TMV RNA molecule. It is assumed that the AO region has a specific tertiary structure, which is maintained by the long-range interactions as well as by interactions of the pseudoknot type.