Context effects: translation of UAG codon by suppressor tRNA is affected by the sequence following UAG in the message

The efficiency of various suppressor tRNAs in reading the UAG amber codon has been measured at 42 sites in the lacI gene. Results indicate that: (1) for all suppressors, efficiency is not an a priori value; rather, it is determined at each site by the specific reading context of the suppressed codon; (2) the degree of sensitivity to context effects differs among suppressors. Most affected is amber suppressor supE (su2), whose activity varies over a 20-fold range depending on context; (3) context effects are produced by residues present at the 3' side of the UAG codon. The most important role appears to be played by the base that is immediately adjacent to the codon. When this base is a purine, the amber codon is suppressed more efficiently than when a pyrimidine is in the same position. Superimposed on this initial pattern, the influence of bases further downstream to the UAG triplet can be detected also. The possibility is discussed that context effects are produced by the whole codon following UAG in the message.     

Nucleotide sequences and functional characterization of two tobacco UAG suppressor tRNAGln isoacceptors and their genes

We isolated and sequenced the two major tRNAGln isoacceptors with CUG and UmUG anticodons from the cytoplasm of Nicotiana rustica. These are the first tRNAsGln of nuclear origin characterized in plants. The tRNAGln sequences were used to design probes for the isolation of the corresponding genes from a nuclear DNA library of N. rustica. The two cloned Nicotiana tRNAGln genes, coding for either of the two isoacceptors, are efficiently transcribed in HeLa cell nuclear extract. In vitro translation in the presence of purified Nicotiana tRNAsGln was carried out in a wheat germ extract partially depleted of endogenous tRNAs. Cytoplasmic (cyt) tRNAGlnCUG and to a lesser extent cyt tRNAGlnUmUG stimulated readthrough over the UAG stop codon present in the tobacco mosaic virus-specific context. The two tRNAGln isoacceptors are the second class of natural UAG suppressors identified in plants, in addition to cyt tRNATyrG A which has previously been characterized as the first natural UAG suppressor.     

Defined set of cloned termination suppressors: in vivo activity of isogenetic UAG, UAA, and UGA suppressor tRNAs.

We have cloned an isogenetic set of UAG, UAA, and UGA suppressors. These include the Su7 -UAG, Su7 -UAA, and Su7 -UGA suppressors derived from base substitutions in the anticodon of Escherichia coli tRNATrp and also Su9 , a UGA suppressor derived from a base substitution in the D-arm of the same tRNA. These genes are cloned on high-copy-number plasmids under lac promoter control. The construction of the Su7 -UAG plasmid and the wild-type trpT plasmid have been previously described ( Yarus , et al., Proc. Natl. Acad. Sci. U.S.A. 77:5092-5097, 1980). Su7 -UAA ( trpT177 ) is a weak suppressor which recognizes both UAA and UAG nonsense codons and probably inserts glutamine. Su7 -UGA ( trpT176 ) is a strong UGA suppressor which may insert tryptophan. Su9 ( trpT178 ) is a moderately strong UGA suppressor which also recognizes UGG (Trp) codons, and it inserts tryptophan. The construction of these plasmids is detailed within. Data on the DNA sequences of these trpT alleles and on amino acid specificity of the suppressors are presented. The efficiency of the cloned suppressors at certain nonsense mutations has been measured and is discussed with respect to the context of these codons.     

Eukaryotic release factor 1 (eRF1) abolishes readthrough and competes with suppressor tRNAs at all three termination codons in messenger RNA.

It is known from experiments with bacteria and eukaryotic viruses that readthrough of termination codons located within the open reading frame (ORF) of mRNAs depends on the availability of suppressor tRNA(s) and the efficiency of termination in cells. Consequently, the yield of readthrough products can be used as a measure of the activity of polypeptide chain release factor(s) (RF), key components of the translation termination machinery. Readthrough of the UAG codon located at the end of the ORF encoding the coat protein of beet necrotic yellow vein furovirus is required for virus replication. Constructs harbouring this suppressible UAG codon and derivatives containing a UGA or UAA codon in place of the UAG codon have been used in translation experiments in vitro in the absence or presence of human suppressor tRNAs. Readthrough can be virtually abolished by addition of bacterially-expressed eukaryotic RF1 (eRF1). Thus, eRF1 is functional towards all three termination codons located in a natural mRNA and efficiently competes in vitro with endogenous and exogenous suppressor tRNA(s) at the ribosomal A site. These results are consistent with a crucial role of eRF1 in translation termination and forms the essence of an in vitro assay for RF activity based on the abolishment of readthrough by eRF1.     

The molecular basis for the differential translation of TMV RNA in tobacco protoplasts and wheat germ extracts

Translation of tobacco mosaic virus (TMV) RNA in tobacco protoplasts yields the 17.5-K coat protein, a 126-K protein and a 183-K protein which is generated by an efficient readthrough over the UAG termination codon at the end of the 126-K cistron. In wheat germ extracts, however, only the 5'-proximal 126-K cistron is translated whereas the 183-K readthrough protein is not synthesized. Purification and sequence analysis of the endogenous tyrosine tRNAs revealed that the uninfected tobacco plant contains two tRNAs^Tyr, both with GΨA anticodons which stimulate the UAG readthrough in vitro and presumably in vivo. In contrast, ˜85% of the tRNA^Tyr from wheat germ contains a QΨA anticodon and ˜15% has a GΨA anticodon. Otherwise the sequences of tRNAs^Tyr from wheat germ and tobacco are identical. UAG readthrough and hence synthesis of the 183-K protein is only stimulated by tRNA^Tyr_GΨA and not at all by tRNA^Tyr_QΨA. The tRNAs^Tyr from wheat leaves were also sequenced. This revealed that adult wheat contains tRNA^Tyr_GΨA only. This is very much in contrast to the situation in animals, where Q-containing tRNAs are characteristic for adult tissues whereas Q deficiency is typical for the neoplastic and embryonic state.     

Transglutaminase activity changes during the hypersensitive reaction, a typical defense response of tobacco NN plants to TMV

The occurrence of glutamyl polyamines (PAs) and changes in activity and levels of transglutaminase (TGase, EC 2.3.2.13), the enzyme responsible for their synthesis, are reported during the progression of the hypersensitive reaction (HR) of resistant NN tobacco plants ( Nicotiana tabacum L. cv. Samsun) to tobacco mosaic virus (TMV). Mature leaves of tobacco were collected over 0–72 h after inoculation with TMV or phosphate buffer (mock). In vivo synthesis of polyamine glutamyl derivatives (glutamyl PAs), catalyzed by TGase activity, was evaluated after supplying labeled putrescine (Pu, a physiological substrate of TGase) to leaves. Results show that, starting from 24 h, mono-( γ -glutamyl)-Pu and bis-( γ -glutamyl)-Sd were recovered in TMV-inoculated samples but not in mock-inoculated ones; 2 days later, in the former, the amount of glutamyl derivatives further increased. An in vitro radiometric assay showed that, in TMV-inoculated leaves, TGase activity increased from 24 h onwards relative to mock controls. An immunoblot analysis with AtPng1p polyclonal antibody detected a 72-kDa protein whose amount increased at 72 h in TMV-inoculated leaves and in the lesion-enriched areas. A biotin-labeled cadaverine incorporation assay showed that TGase activity occurred in S1 (containing soluble proteins), S2 (proteins released by both cell walls and membranes) and S3 (membrane intrinsic proteins) fractions. In S3 fraction, where changes were the most relevant, TGase activity was enhanced in both mock-inoculated and TMV-inoculated samples, but the stimulation persisted only in the latter case. These data are discussed in the light of a possible role of TGase activity and glutamyl PAs in the defense against a viral plant pathogen.     

Effects of two TMV strains on the synthesis and stability of chloroplast ribosomal RNA in tobacco leaves

Summary Multiplication of TMV-strains vulgare (light-green/dark-green mosaic symptoms) and flavum (severe yellow/green mosaic) had different effects on the ribosomal RNA of tobacco leaf chloroplasts. Vulgare inhibited chloroplast ribosomal RNA synthesis while having no effect on cytoplasmic ribosomal RNA synthesis (Fig. 2). Flavum inhibited chloroplast ribosomal RNA synthesis more severely than vulgare, and caused an earlier degradation of chloroplast ribosomal RNA than in control or vulgare-infected leaves (Fig. 1). Flavum also inhibited cytoplasmic ribosomal RNA synthesis. A connection between these differing effects on chloroplast ribosomal RNA metabolism and severity of visible symptoms is suggested, and discussed in relation to a possible influence on symptoms of denatured virus coat protein.Abbreviations TMV Tobacco Mosaic Virus - RNA Ribonucleic acid - DNA Deoxyribonucleic acid - m millions (in molecular weight values)     

Changes in key enzymes of viral-RNA biosynthesis in chloroplasts from PVY and TMV infected tobacco plants

Effects of the infection with tobacco mosaic virus (TMV) and potato virus Y (PVY) on chloroplasts from susceptible tobacco plants were determined. Changes in ribonucleases (RNases), phosphomonoesterase (PME), phosphodiesterase (PDE), glucose-6-phosphate dehydrogenase (G6P DH), 6-phosphogluconate dehydrogenase (6PG DH), glucokinase (GK), and fructokinase (FK) activities in thylakoid/envelope and stroma fractions were studied. Slight increase in the activities of PME, PDE, G6P DH and 6PG DH of thylakoid/envelope fraction as well as of RNases, PME, PDE, G6P DH, 6PG DH, GK and FK of stroma fraction was found in chloroplasts isolated from leaf tissues infected with PVY. Infection with TMV produced higher increase in enzymes activities in chloroplasts; especially, PME, G6P DH and 6PG DH in fraction of thylakoid/envelope, and RNases, PME, PDE, G6P DH, 6PG DH, and GK in stroma fraction.This study was supported by grant No. 522/02/0708 of the Grant Agency of the Czech Republic.     

RNA isolation from loquat and other recalcitrant woody plants with high quality and yield

RNA isolation is difficult in plants that contain large amounts of polysaccharides and polyphenol compounds. To date, no commercial kit has been developed for the isolation of high-quality RNA from tissues with these characteristics, especially for fruit. The common protocols for RNA isolation are tedious and usually result in poor yields when applied to recalcitrant plant tissues. Here an efficient RNA isolation protocol based on cetyltrimethylammonium bromide (CTAB) and two successive precipitations with 10 M lithium chloride (LiCl) was developed specifically for loquat fruits, but it was proved to work efficiently in other tissues of loquat and woody plants. The RNA isolated by this improved protocol was not only of high purity and integrity (A₂₆₀/A₂₈₀ ratios ranged from 1.90 to 2.04 and A₂₆₀/A₂₃₀ ratios were > 2.0) but also of high yield (up to 720 μg on average [coefficient of variation = 21%] total RNA per gram fresh tissue). The protocol was tested on loquat fruit (different stages of development, postharvest, ripening, and bruising), leaf, root, flower, stem, and bud; quince fruit and root; grapevine cells in liquid culture; and rose petals. The RNA obtained with this method is amenable to enzymatic treatments and can be efficiently applied for research on gene characterization, expression, and function.     

UAG readthrough in mammalian cells: Effect of upstream and downstream stop codon contexts reveal different signals

Background  

Translation termination is mediated through an interaction between the release factors eRF1 and eRF3 and the stop codon within its nucleotide context. Although it is well known that the nucleotide contexts both upstream and downstream of the stop codon, can modulate readthrough, little is known about the mechanisms involved.     

Short double-stranded RNA with immunostimulatory activity: sequence dependence

Small interfering RNAs (siRNA) are able to activate the mammalian innate immune system depending on their structure, sequence, and method of delivery. The immunostimulatory activity of double-stranded RNA can be applied to antiviral and antitumor therapy. Here we identified a set of 19-bp RNA duplexes with 3-nucleotid overhangs in the 3' ends that display immunostimulating activity (here and after immunostimulating RNA, or isRNA) and studied their sequence/activity relationships. It was found that the introduction of substitutions in the middle part of the isRNA sequence (10-16 positions counting from the 5' end of strand 1) does not alter the antiproliferative activity, while substitutions in the 3' end region of isRNA substantially reduce it. isRNAs efficiently inhibit the proliferation of human oral epidermoid carcinoma cells [half-maximal inhibitory concentration (IC(50)) values varied from 10 to 100 nM]. Our research demonstrated that antiproliferative effects of isRNAs are related to cell growth arrest, rather than the induction of apoptosis. These isRNAs strongly stimulate the synthesis of interferon-α (IFN-α), and to a lesser extent the synthesis of tumor necrosis factor (TNF-α) and interleukin-6 (IL-6), in adherent peripheral blood mononuclear cells. An intravenous injection of isRNA/Lipofectamine complexes into C57BL mice increases IFN-α and IL-6 levels in the blood serum up to 15-fold and 3-fold, respectively, compared to the control mice. The results obtained clearly demonstrate the pronounced immunostimulatory and antiproliferative properties of the isRNAs under study. Hence, these short double-stranded RNAs can be considered as potential agents for the therapy of oncological and viral diseases.     

Mutational alterations of tryptophan-specific transfer RNA that generate translation suppressors of the UAA, UAG and UGA nonsense codons

The recessive lethal amber suppressor su⁺7(UAG-1) in Escherichia coli inserts glutamine in response to the UAG codon. The genetic analysis presented in this paper shows that the su^?7 precursor allele can give rise to suppressors of the UGA codon as well as of the UAG codon. This observation suggests that the su^?7 gene normally codes for transfer RNA^Trp, a tRNA whose anticodon can be modified by single base changes to forms that can translate either UAG or UGA. The chemical findings presented in the accompanying paper (Yaniv et al., 1974) are wholly in accord with this interpretation. Thus, a single base substitution in the anticodon sequence of a tRNA can affect both the coding specificity of the molecule and also the amino acid acceptor specificity.     

A single mutational modification of a tryptophan-specific transfer RNA permits aminoacylation by glutamine and translation of the codon UAG

In this work we show that the wild-type (su^?7) progenitor of the recessivelethal suppressors of UAG (su⁺7(UAG)) and of UAA/G (su⁺7(UAA/G)) is the structural gene for transfer RNA^Trp, the adaptor for translating the codon UGG. The su⁺7(UAG) suppressor form of the tRNA has a C for U substitution in the middle base of the anticodon; in the su⁺7(UAA/G) suppressor tRNA both C residues of the anticodon are replaced by U. Our data establish that the mutational change altering the tRNA^Trp to a UAG suppressor is accompanied by a loss of tryptophan-accepting specificity and the acquisition of glutamine-acceptor activity.     

The nucleotide sequence of 4.5S ribosomal RNA from tobacco chloroplasts.

The nucleotide sequence of tobacco chloroplast 4.5S ribosomal RNA has been determined to be: OHG-A-A-G-G-U-C-A-C-G-G-C-G-A-G-A-C-G-A-G-C-C-G-U-U-U-A-U-C-A-U-U-A-C-G-A-U-A-G-G-U-G-U-C-A-A-G-U-G-G-A-A-G-U-G-C-A-G-U-G-A-U-G-U-A-U-G-C-(G-A)-C-U-G-A-G-G-C-A-U-C-C-U-A-A-C-A-G-A-C-C-G-G-U-A-G-A-C-U-U-G-A-A-COH. The 4.5S RNA is 103 nucleotides long and its 5'-terminus is not phosphorylated.     

Dramatic events in ciliate evolution: alteration of UAA and UAG termination codons to glutamine codons due to anticodon mutations in two Tetrahymena tRNAs

The three major glutamine tRNAs of Tetrahymena thermophila were isolated and their nucleotide sequences determined by post-labeling techniques. Two of these tRNAs^Gln show unusual codon recognition: a previously isolated tRNA^Gln_UmUA and a second species with CUA in the anticodon (tRNA^Gln_CUA). These two tRNAs recognize two of the three termination codons on natural mRNAs in a reticulocyte system. tRNA^Gln_UmUA reads the UAA codon of α-globin mRNA and the UAG codon of tobacco mosaic virus (TMV) RNA, whereas tRNA^Gln_CUA recognizes only UAG. This indicates that Tetrahymena uses UAA and UAG as glutamine codons and that UGA may be the only functional termination codon. A notable feature of these two tRNAs^Gln is their unusually strong readthrough efficiency, e.g. purified tRNA^Gln_CUA achieves complete readthrough over the UAG stop codon of TMV RNA. The third major tRNA^Gln of Tetrahymena has a UmUG anticodon and presumably reads the two normal glutamine codons CAA and CAG. The sequence homology between tRNA^Gln_UmUG and tRNA^Gln_UmUA is 81%, whereas that between tRNA^Gln_CUA and tRNA^Gln_UmUA is 95%, indicating that the two unusual tRNAs^Gln evolved from the normal tRNA^Gln early in ciliate evolution. Possible events leading to an altered genetic code in ciliates are discussed.     

cDNA cloning of six mRNAs induced by TMV infection of tobacco and a characterization of their translation products

A cDNA library was constructed to 10-15 S poly(A) RNA from tobacco mosaic virus (TMV)-infected Samsun NN tobacco. By differential colony hybridization of 1400 transformants, 32 clones were obtained corresponding to TMV-inducible tobacco mRNAs. These clones were subdivided into six clusters on the basis of cross-hybridization of the inserts. By Northern blot hybridization it was shown that three of the corresponding mRNAs were strongly induced by spraying tobacco plants with salicylic acid, whereas one mRNA was weakly induced by this treatment. All mRNAs were systemically induced in plants in which only the lower leaves were locally infected by TMV. Hybrid-selected translation was performed, using six clones representing one cluster each, followed by immunoprecipitation using an antiserum to purified pathogenesis-related (PR) proteins. Four clones yielded precipitable translation products. One of these clones represented a cluster of PR-1 clones, another clone encoded the thaumatin-like (TL) protein of tobacco which may correspond to PR-P or −Q.