Tobacco rattle virus from a Hosta hybrid contained one RNA1 (Ho-1) and two RNA2 species (Ho-2a, Ho-2b). Whereas Ho-1 resembles TRV Al RNA1 from Alstroemerias, Ho-2a and Ho-2b resemble TRV TpO1 RNA2... Show moreTobacco rattle virus from a Hosta hybrid contained one RNA1 (Ho-1) and two RNA2 species (Ho-2a, Ho-2b). Whereas Ho-1 resembles TRV Al RNA1 from Alstroemerias, Ho-2a and Ho-2b resemble TRV TpO1 RNA2 from a potato field. Ho-2a has a complete RNA2-specific sequence, whereas that of Ho2-b carries a large deletion. The short RNA1-related 3' end of Ho-2a is distinct from that of Ho-1, whereas the longer one of Ho-2b is identical to that of Ho-1. TRV RNA2 molecules may apparently become associated with different TRV RNA1 molecules, from which they can acquire 3'ends of various lengths while often losing large portions of their RNA2-specific sequences. Show less
Nowadays, the development of experimental procedures for the determination of the secondary structure of RNA molecules is taking advantage of the novel single-molecule probing and imaging... Show moreNowadays, the development of experimental procedures for the determination of the secondary structure of RNA molecules is taking advantage of the novel single-molecule probing and imaging techniques. We report a method for the mapping of the secondary structure of RNA molecules spread on a flat surface by means of the atomic force microscope. Globular domains comprising groups of RNA secondary and tertiary structure elements separated by unstructured domains can be discerned in the micrographs and their position along the molecule contour can be measured directly on unstained specimens. We have analyzed the morphology of a population of single molecules of 3' fragments of the Turnip Yellow Mosaic Virus RNA shorter than 1 kb in different temperature and electrolytic conditions. We found a satisfying agreement of the shape of the imaged structures with previously available evidence. The method we have developed can be used to map also different types of RNA molecules and has the advantage of showing the distribution of the single molecule conformations within the population. (C) 2005 Wiley-Liss, Inc. Show less
RNA-coat protein interactions in turnip yellow mosaic virus (TYMV) have been shown to involve low pK proton-donating groups. Two different types of interaction have been proposed. In the so-called... Show moreRNA-coat protein interactions in turnip yellow mosaic virus (TYMV) have been shown to involve low pK proton-donating groups. Two different types of interaction have been proposed. In the so-called type I interaction, protonated C-residues interact with acidic amino acids at low pH, thereby providing a rationale for the high C-content (38%) of the genomic RNA. The type 11 interaction involves charged histidines interacting with phosphates of the RNA backbone. Site-directed mutagenesis of the TYMV coat protein and subsequent in vivo analysis were performed to distinguish between these two types of RNA-protein interaction. The results reveal a prominent role for the histidines H68 and H180, since mutation to an alanine residue inhibits symptom development on secondary leaves, indicating that spreading of the virus in the plant is blocked. Viral RNA and coat protein synthesis are not altered, showing that these two histidines may play a role in the process of RNA encapsidation. Overexpression of the TYMV coat protein in Escherichia coli leads to the formation of bona fide capsids, showing that the two histidines are not critical in capsid assembly. Mutagenesis of the acidic amino acids D11, E135, and D143 to alanine apparently did not interfere with virus viability. The functional role of the histidines during the infection cycle is discussed in terms of the structure of the coat protein, both at the level of amino acid sequence conservation among the members of the Tymoviridae family and as the three-dimensional structure of the coat protein. (C) 2004 Wiley-Liss, Inc. Show less
For various groups of plant viruses, the genomic RNAs end with a IRNA-like structure (TLS) instead of the 3' poly(A) tail of common mRNAs. The actual function of these TLSs has long been enigmatic.... Show moreFor various groups of plant viruses, the genomic RNAs end with a IRNA-like structure (TLS) instead of the 3' poly(A) tail of common mRNAs. The actual function of these TLSs has long been enigmatic. Recently, however, it became clear that for turnip yellow mosaic virus, a tymovirus, the valylated TLSTYMV of the single genomic RNA functions as a bait for host ribosomes and directs them to the internal initiation site of translation (with N-terminal valine) of the second open reading frame for the polyprotein. This discovery prompted us to investigate whether the much larger TLSs of a different genus of viruses have a comparable function in translation. Brome mosaic virus (BMV), a bromovirus, has a tripartite RNA genome with a subgenomic RNA4 for coat protein expression. All four RNAs carry a highly conserved and bulky 3' TLSBMV (about 200 nucleotides) with determinants for tyrosylation. We discovered TLSBMV-catalyzed self-tyrosylation of the tyrosyl-tRNA synthetase but could not clearly detect tyrosine incorporation into any virus-encoded protein. We established that BMV proteins do not need TLSBMV tyrosylation for their initiation. However, disruption of the TLSs strongly reduced the translation of genomic RNA1, RNA2, and less strongly, RNA3, whereas coat protein expression from RNA4 remained unaffected. This aberrant translation could be partially restored by providing the TLSBMV in trans. Intriguingly, a subdomain of the TLSBMV could even almost fully restore translation to the original pattern. We discuss here a model with a central and dominant role for the TLSBMV during the BMV infection cycle. Show less
The 5' untranslated region (UTR) of the RNA of several tymoviruses contains conserved hairpins with protonatable internal loops, consisting of C-C and C-A mismatches (K. Hellendoorn, P. J. A.... Show moreThe 5' untranslated region (UTR) of the RNA of several tymoviruses contains conserved hairpins with protonatable internal loops, consisting of C-C and C-A mismatches (K. Hellendoorn, P. J. A. Michiels, R. Buitenhuis, and C. W. A. Pleij, Nucleic Acids Res. 24, 4910-4917, 1996). Here, we present a functional analysis of the 5' UTR of turnip yellow mosaic virus (TYMV) RNA, which contains two protonatable hairpins with nearly identical internal loops, Mutations were introduced in an infectious cDNA clone, and T7 RNA transcripts were used to infect Chinese cabbage plants, Different symptoms were observed for the various mutants, pointing to a functional role of the C-C and C-A mismatches in the hairpins of the 5' UTR. The replication of the virus is influenced by the mutations made, while in vitro translation studies showed that the expression of the two overlapping reading frames of TYMV is not influenced by the secondary structure of the leader. Various mutants were propagated for up to five serial passages of infection, and the sequence of the 5' UTR was determined. This resulted in virus RNA with new non-wild-type sequences that produced the wild-type phenotype in infected plants, Remarkably, in all cases C-C or C-A mismatches were introduced. The internal loop of the 5'-proximal hairpin seems to be more important for the viral life cycle than that of the second hairpin, A deletion of 75% of the leader, including the two hairpins, resulted in a virus that was deficient in viral spread. Since the ratio between filled and empty capsids was changed drastically by this mutation, a role of the 5' UTR in viral packaging is proposed. Show less
The tRNA-like structure at the 3' end of turnip yellow mosaic virus (TYMV) RNA was studied in order to determine the role of this structure in the initiation of minus-strand synthesis in vitro.... Show moreThe tRNA-like structure at the 3' end of turnip yellow mosaic virus (TYMV) RNA was studied in order to determine the role of this structure in the initiation of minus-strand synthesis in vitro. Deletions in the 5'-to 3' direction up to the pseudoknot structure did not result in a decrease of transcription efficiency. However, transcription efficiency was reduced twofold when a fragment of 21 nucleotides, comprising the 3'-terminal hairpin, was used as a template, tRNA(Phe) from yeast, Escherichia coli 5S rRNA, and the 3'-terminal 208 nucleotides of alfalfa mosaic virus RNA 3 could not be transcribed by the RNA-dependent RNA polymerase (RdRp) of TYMV. Various mutations in the sequences of loop regions L1 and L2 or of stem region S1 of the pseudoknot were tested to further investigate the importance of the pseudoknot structure. The results were compared with those obtained in an earlier study on aminoacylation with the same mutants tR, M. W. Mans, M. H. van Steeg, P. W. G. Verlaan, C. W. A. Pleij, and L. Bosch. J. Mol. Biol, 223:221-232: 1992). Mutants which still harbor a stable pseudoknot, as proven by probing its structure, have a transcription efficiency very close to that of the wild-type virus. Disruption of the pseudoknot structure, however, gives rise to a drop in transcription efficiency to about 50%, No indications of base-specific interactions between L1, L2, or S1 of the pseudoknot and the RdRp were found. Show less
We report that tyro subtypes of alpha(2)-adrenergic receptors (alpha(2A/D)- and alpha(2C)-AR) are ectopically expressed with dramatically different efficiencies and that this difference is due to a... Show moreWe report that tyro subtypes of alpha(2)-adrenergic receptors (alpha(2A/D)- and alpha(2C)-AR) are ectopically expressed with dramatically different efficiencies and that this difference is due to a 288-nucleotide (nt) segment in the 3'-untranslated region (3'-UTR) of the alpha(2C)-AR mRNA that impairs translational processing, NIH-3T3 fibroblasts mere transfected with receptor constructs (coding region plus 552 nt, alpha(2C)-AR; coding region plus 1140 nt, alpha(2A/D)-AR) and a vector conferring G418 resistance. Transcription was driven by the murine sarcoma virus promoter element, and the receptor gene segment was upstream of an SV40 polyadenylation cassette. Drug-resistant transfectants were evaluated for expression of receptor mRNA and protein, 90% of the NIH-3T3 alpha(2C)-AR transfectants expressed receptor mRNA, but only 14% of the clonal cell Lines expressed receptor protein. In contrast, 90% of the NIH-3T3 alpha(2A/D)-AR transfectants expressed receptor protein (200-5000 fmol/mg). Similar results were obtained following transfection of DDT1MF-2 cells with the two receptor constructs. The role of the 3'-UTR of the alpha(2C)-AR in mRNA processing was determined by generating new constructs in which the 3'-UTR. was progressively truncated from 552 to 470, 182, 143, or 74 nt 3' to the stop codon. Truncation of the 3'-UTR resulted in the expression of receptor protein in the G418-resistant transfectants (nt 74, 100%; nt 143, 80%; nt 182, 50%). The level of mRNA in the transfectants expressing the receptor protein was not greater than that in nonexpressing clones, and the differences in protein expression did not reflect altered mRNA stability in the truncated construct. The alpha(2C)-AR mRNA with the longer 3'-UTR underwent translational initiation as it was found in the polysome fraction, indicating that the lack of receptor protein was due to impaired translational elongation or termination. These data suggest that translational efficiency is a hey mechanism for regulating alpha(2C)-AR expression and associated signaling events. Show less
The RNA-dependent RNA polymerase (RdRp) of turnip yellow mosaic virus (TYMV) was isolated by a simple, new method. An active, template-dependent and specific enzyme was obtained. Although the... Show moreThe RNA-dependent RNA polymerase (RdRp) of turnip yellow mosaic virus (TYMV) was isolated by a simple, new method. An active, template-dependent and specific enzyme was obtained. Although the genomic RNA of TYMV could not be transcribed completely during an in vitro RdRp assay, a complete double-stranded product was obtained when a 3' terminal RNA fragment of 83 nucleotides was used as a template. The reaction product was identified as being of negative polarity by complete digestion With ribonuclease T1. Antibodies directed to part of the N-terminal (Ab140) or C-terminal (Ab66) in vitro autocleavage products of the large non-structural polyprotein of TYMV, could both partially inhibit RdRp activity. Further purification of the RdRp preparation by ion-exchange chromatography resulted in two activity peaks with different protein compositions. Both peak fractions retained high specificity for transcription of TYMV RNA. A protein of approximately 115 kDa was detected by both Ab140 and Ab66. (C) 1997 Elsevier Science B.V. Show less
Poot, R.A.; Jeeninga, R.E.; Pleij, C.W.A.; Duin, J. van 1997
We have analyzed the ribosomal protein profile of Escherichia coli 30S subunits with the mutation C(18)A in the central pseudoknot of their 16S ribosomal RNA, This mutation was shown to inhibit... Show moreWe have analyzed the ribosomal protein profile of Escherichia coli 30S subunits with the mutation C(18)A in the central pseudoknot of their 16S ribosomal RNA, This mutation was shown to inhibit translational activity in vivo and to affect ribosome stability in vitro, The majority of the mutant 30S particles were present as free subunits in which a reproducible decrease in amount of proteins S1, S2, S18 and S21 was observed, The protein gels also showed the appearance of a satellite band nest to S5, This band reacted with anti-S5 antibodies and had a slightly increased positive charge, The simplest interpretation of these findings, also considering published data, is that the satellite band is S5 with a non-acetylated N-terminal alanine, Underacetylation of S5 due to mutations in the 16S rRNA implies that the modification is performed on the ribosome. Show less
The pseudoknot or "base-paired loop region" is a widespread structural motif in all kinds of viral RNAs. Detailed structures of hairpin-type pseudoknots, obtained by NMR, are now emerging, but it... Show moreThe pseudoknot or "base-paired loop region" is a widespread structural motif in all kinds of viral RNAs. Detailed structures of hairpin-type pseudoknots, obtained by NMR, are now emerging, but it is still not clear which structural features are responsible for the different functions in processes like translation and replication. Especially noncoding regions are rich sources of pseudoknot structures, where they occur in domains like IRES elements and tRNA-like structures. But also its role in coding regions like in ribosomal -1 frameshifting and read-through is well established, although the precise mechanism of interference with the translational mechanism remains unknown. (C) 1997 Academic Press. Show less
