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PubMed1998

Nucleotide sequences and functional characterization of two tobacco UAG suppressor tRNA(Gln) isoacceptors and their genes.

We isolated and sequenced the two major tRNA(Gln) isoacceptors with CUG and UmUG anticodons from the cytoplasm of Nicotiana rustica. These are the first tRNAs(Gln) of nuclear origin characterized in plants. The tRNA(Gln) 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 tRNA(Gln) 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 tRNAs(Gln) was carried out in a wheat germ extract partially depleted of endogenous tRNAs. Cytoplasmic (cyt) tRNA(Gln)CUG and to a lesser extent cyt tRNA(Gln)UmUG stimulated readthrough over the UAG stop codon present in the tobacco mosaic virus-specific context. The two tRNA(Gln) isoacceptors are the second class of natural UAG suppressors identified in plants, in addition to cyt tRNA(Tyr)GpsiA which has previously been characterized as the first natural UAG suppressor.

Anticodon↗
PubMed2005

A newly discovered function for RNase L in regulating translation termination.

The antiviral and antiproliferative effects of interferons are mediated in part by the 2'-5' oligoadenylate-RNase L RNA decay pathway. RNase L is an endoribonuclease that requires 2'-5' oligoadenylates to cleave single-stranded RNA. In this report we present evidence demonstrating a role for RNase L in translation. We identify and characterize the human translation termination factor eRF3/GSPT1 as an interacting partner of RNase L. We show that interaction of eRF3 with RNase L leads to both increased translation readthrough efficiency at premature termination codons and increased +1 frameshift efficiency at the antizyme +1 frameshift site. On the basis of our results, we present a model describing how RNase L is involved in regulating gene expression by modulating the translation termination process.

Carrier Proteins↗
PubMedDate not supplied

The origin of the molecular diversity and functional anchoring of cholinesterases.

Vertebrates possess two cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) which both hydrolyze acetylcholine, but differ in their specificity towards other substrates, and in their sensitivity to inhibitors. In mammals, the AChE gene produces three types of coding regions through the choice of 3' splice acceptor sites, generating proteins which possess the same catalytic domain, associated with distinct C-terminal peptides. AChE subunits of type R ('readthrough') produce soluble monomers; they are expressed during development and induced by stress in the mouse brain. AChE subunits of type H ('hydrophobic') produce GPI-anchored dimers, but also secreted molecules; they are mostly expressed in blood cells. Subunits of type T ('tailed') exist for both AChE and BChE. They represent the enzyme forms expressed in brain and muscle. These subunits generate a variety of quaternary structures, including homomeric oligomers (monomers, dimers, tetramers), as well as hetero-oligomeric assemblies with anchoring proteins, ColQ and PRiMA. Mutations in the four-helix bundle (FHB) zone of the catalytic domain indicate that subunits of type H and T use the same interaction for dimerization. On the other hand, the C-terminal T peptide is necessary for tetramerization. Four T peptides, organized as amphiphilic alpha helices, can assemble around proline-rich motifs of ColQ or PRiMA. The association of AChE(T) or BChE subunits with ColQ produces collagen-tailed molecules, which are inserted in the extracellular matrix, e.g. in the basal lamina of neuromuscular junctions. Their association with PRiMA produces membrane-bound tetramers which constitute the predominant form of cholinesterases in the mammalian brain; in muscles, the level of PRiMA-anchored tetramers is regulated by exercise, but their functional significance remains unknown. In brain and muscles, the hydrolysis of acetylcholine by cholinesterases, in different contexts, and their possible noncatalytic functions clearly depend on their localization by ColQ or PRiMA.

Acetylcholinesterase↗
PubMed2002

Rous sarcoma virus negative regulator of splicing selectively suppresses SRC mRNA splicing and promotes polyadenylation.

Retroviruses require a balance of spliced and unspliced RNA for efficient replication. Here, we examined the effect of mutations in a splicing suppressor sequence called the negative regulator of splicing (NRS), located within the gag gene of Rous sarcoma virus. While the NRS mutant viruses showed only small changes in the levels of spliced env mRNAs, they had significant increases in src mRNA levels and transformed cells more efficiently than wild-type virus. None of these mutations prevented viral replication; however, some of the mutant viruses replicated more slowly than wild-type virus. In addition, increased transcriptional readthrough of the poly(A) site in the 3' LTR was observed with the NRS mutant viruses, suggesting that the wild-type NRS sequence promotes polyadenylation.

Animals↗
PubMed2006

Effects of an opal termination codon preceding the nsP4 gene sequence in the O'Nyong-Nyong virus genome on Anopheles gambiae infectivity.

The genomic RNA of an alphavirus encodes four different nonstructural proteins, nsP1, nsP2, nsP3, and nsP4. The polyprotein P123 is produced when translation terminates at an opal termination codon between nsP3 and nsP4. The polyprotein P1234 is produced when translational readthrough occurs or when the opal termination codon has been replaced by a sense codon in the alphavirus genome. Evolutionary pressures appear to have maintained genomic sequences encoding both a stop codon (opal) and an open reading frame (arginine) as a general feature of the O'nyong-nyong virus (ONNV) genome, indicating that both are required at some point. Alternate replication of ONNVs in both vertebrate and invertebrate hosts may determine predominance of a particular codon at this locus in the viral quasispecies. However, no systematic study has previously tested this hypothesis in whole animals. We report here the results of the first study to investigate in a natural mosquito host the functional significance of the opal stop codon in an alphavirus genome. We used a full-length cDNA clone of ONNV to construct a series of mutants in which the arginine between nsP3 and nsP4 was replaced with an opal, ochre, or amber stop codon. The presence of an opal stop codon upstream of nsP4 nearly doubled (75.5%) the infectivity of ONNV over that of virus possessing a codon for the amino acid arginine at the corresponding position (39.8%). Although the frequency with which the opal virus disseminated from the mosquito midgut did not differ significantly from that of the arginine virus on days 8 and 10, dissemination did began earlier in mosquitoes infected with the opal virus. Although a clear fitness advantage is provided to ONNV by the presence of an opal codon between nsP3 and nsP4 in Anopheles gambiae, sequence analysis of ONNV RNA extracted from mosquito bodies and heads indicated codon usage at this position corresponded with that of the virus administered in the blood meal. These results suggest that while selection of ONNV variants is occurring, de novo mutation at the position between nsP3 and nsP4 does not readily occur in the mosquito. Taken together, these results suggest that the primary fitness advantage provided to ONNV by the presence of an opal codon between nsP3 and nsP4 is related to mosquito infectivity.

Alphavirus↗
PubMed2005

DEG1, encoding the tRNA:pseudouridine synthase Pus3p, impacts HOT1-stimulated recombination in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, HOT1-stimulated recombination has been implicated in maintaining homology between repeated ribosomal RNA genes. The ability of HOT1 to stimulate genetic exchange requires RNA polymerase I transcription across the recombining sequences. The trans-acting nuclear mutation hrm3-1 specifically reduces HOT1-dependent recombination and prevents cell growth at 37 degrees . The HRM3 gene is identical to DEG1. Excisive, but not gene replacement, recombination is reduced in HOT1-adjacent sequences in deg1Delta mutants. Excisive recombination within the genomic rDNA repeats is also decreased. The hypo-recombination and temperature-sensitive phenotypes of deg1Delta mutants are recessive. Deletion of DEG1 did not affect the rate of transcription from HOT1 or rDNA suggesting that while transcription is necessary it is not sufficient for HOT1 activity. Pseudouridine synthase 3 (Pus3p), the DEG1 gene product, modifies the anticodon arm of transfer RNA at positions 38 and 39 by catalyzing the conversion of uridine to pseudouridine. Cells deficient in pseudouridine synthases encoded by PUS1, PUS2 or PUS4 displayed no recombination defects, indicating that Pus3p plays a specific role in HOT1 activity. Pus3p is unique in its ability to modulate frameshifting and readthrough events during translation, and this aspect of its activity may be responsible for HOT1 recombination phenotypes observed in deg1 mutants.

Alleles↗
PubMed1998

Transcription and transcript processing in the sdhCDAB-sucABCD operon of Escherichia coli.

The genes encoding succinate dehydrogenase (sdhCDAB), the specific components of the 2-oxoglutarate dehydrogenase complex (ODH, E1o and E2o; sucAB) and succinyl-CoA synthetase (sucCD) form a cluster containing two promoters at 16.3 min in the chromosome of Escherichia coli: Psdh sdhCDAB-Psuc sucAB-sucCD. The gene encoding the lipoamide dehydrogenase component of both the 2-oxoglutarate and pyruvate dehydrogenase complexes (E3; lpdA) is the distal gene of another cluster containing two promoters located at 2.7 min: Ppdh pdhR-aceEF-Plpd lpdA. The responses of the suc and lpd promoters to different environmental conditions and to regulator defects were investigated with appropriate lacZ fusions, in order to understand how expression of the sucAB genes is co-regulated with other genes in the sdhCDAB-sucABCD cluster and with lpdA expression. Expression from the suc promoter was repressed by IHF and partially activated by sigma 38 but it was not regulated by ArcA, FNR, CRP, FruR or Fis, and not repressed by glucose or anaerobiosis, indicating that the well-established catabolite and anaerobic repression of ODH synthesis is imposed elsewhere. In contrast, the lpd promoter was repressed by both glucose (via a CRP-independent mechanism) and anaerobiosis (mediated by ArcA), and activated by Fis, but it was not regulated by FNR, FruR, IHF or sigma 38. These observations support the view that transcription of the sucABCD genes is primarily initiated and regulated at the upstream sdh promoter, and that the lpd promoter is independently co-regulated with Psdh (primarily by ArcA-mediated repression) rather than with Psuc. Direct evidence for co-transcription of the entire sdhCDAB-sucABCD region from Psdh was obtained by detecting a 10 kb transcript in rnc and rne mutants, but not in the parental strains. Three RNaseIII-specific processing sites, which contribute to the extreme instability of the readthrough transcript, were identified in the sdhCDAB-sucABCD intergenic region. Other sites of endonuclease processing were located by interpreting the patterns of transcript subfragments observed in Northern blotting.

Base Sequence↗
PubMed1998

Translational readthrough in the hdc mRNA generates a novel branching inhibitor in the drosophila trachea.

A central question in the development of many branched tubular organs, including the Drosophila trachea, concerns the mechanisms and molecules that control the number and pattern of new branches arising from preexisting vessels. We report on a branching inhibitor, Fusion-6 (Fus-6) produced by specialized tracheal cells to prevent neighboring cells from branching. In Fus-6 mutants, cells that are normally quiescent acquire the branching fate and form an increased number of sprouts emanating from the primary branches. Fus-6 is identified as the headcase (hdc) gene and is expressed in a subset of the cells that extend fusion sprouts to interconnect the tracheal network. hdc expression is regulated by the transcription factor escargot (esg) because it is not expressed in the fusion cells of esg mutants and is ectopically activated in the trachea in response to esg misexpression. We show that the hdc mRNA encodes two overlapping protein products by an unusual suppression of translational termination mechanism. Translational readthrough is necessary for hdc function because rescue of the tracheal mutant phenotype requires the full-length hdc mRNA. In ectopic expression experiments with full-length and truncated hdc constructs, only the full-length cDNA encoding both proteins could inhibit terminal branching. We propose that hdc acts non-autonomously in an inhibitory signaling mechanism to determine the number of cells that will form unicellular sprouts in the trachea.

Amino Acid Sequence↗
PubMed1989

Early transcribed sequences affect termination efficiency of Escherichia coli RNA polymerase.

We have constructed novel transcription templates in which we have fused the late gene promoters of Escherichia coli phages lambda and 82 upstream from three different rho-independent transcription terminators. Using an in vitro transcription assay and an in vivo galactokinase expression assay, we find that the initial portion of the transcribed region significantly affects the efficiency of some downstream terminators. We have identified, by deletion, substitution and point mutation analysis, sequences responsible for these increased levels of factor-independent readthrough. Since these important sequences occur within about 30 nucleotides of the RNA start site, we suggest that the initial portion of the transcript can affect termination efficiency.

Bacteriophages↗
PubMed1987

Mutants of elongation factor Tu promote ribosomal frameshifting and nonsense readthrough.

This is the first report of ribosomal frameshifting promoted by mutants of the elongation factor Tu (EF-Tu). EF-Tu mutants can suppress both -1 and +1 frameshift mutations. The level of nonsense readthrough is also increased at some UGA (this paper) and UAG (Hughes, 1987) sites by these mutants. Suppression occurs when a mutant tuf allele is paired with a wild-type copy of the other tuf gene but is most efficient when both tuf genes are mutant. Frameshifting mediated by the tuf alleles studied, tufA8 and tufB103, is not general; indeed most frameshift mutations are not suppressed. Several possible mechanisms by which mutant EF-Tu may cause frameshifting are discussed.

Alleles↗
PubMed1994

Functional interactions between translation, transcription and ppGpp in growing Escherichia coli.

Strains with a relA mutation together with three different alleles of spoT were used to study the effects of different levels of ppGpp on production time for beta-galactosidase, transcriptional polarity and readthrough of a stop codon by near-cognate tRNA or a suppressor tRNA. The influences of an rpsL(S12) allele and a miaA mutation, together giving decreased efficiency of translation, as well as an rpoB mutation, coding for an altered RNA polymerase, were also investigated. The spoT alleles which give total deficiency for ppGpp, or a level which is increased several-fold (Sarubbi et al. (1988) Mol. Gen. Genet. 213, 214-222), had at the most a marginal effect on the production time for a beta-galactosidase molecule or translational misreading of a nonsense mutation. The efficiency of an amber tRNA suppressor is not affected by ppGpp in strains with an otherwise wildtype translational machinery. These data suggest that ppGpp does not influence directly the translational process in vivo. Instead, ppGpp is found to interfere with transcriptional readthrough in a manner which is dependent on the rpsL224, miaA, as well as the rpoB mutations. Similarly, bacterial growth is affected by ppGpp in a manner which is dependent on properties of both the transcriptional and translational apparatus together. It is suggested that the primary effect of ppGpp is on transcriptional readthrough, but this effect is modified by translational/transcriptional coupling.

DNA-Directed RNA Polymerases↗
PubMed1983

A cluster of leftward, rho-dependent t'J terminators in the J gene of coliphage lambda.

While searching for the N-unresponsive terminator described by Honigman (Gene 13 (1981) 299-309), a 1680-bp DNA fragment from within gene J of bacteriophage lambda was cloned into plasmid pD12 between promoter p'R and the galK gene. In vitro transcription of this plasmid and S1 mapping assays, together with nucleotide sequencing, demonstrated that this DNA fragment contains a cluster of at least four in rho+ Escherichia coli hosts and only 30% in rho- hosts at at 30 degrees C. At the elevated temperature of 42 degrees C, the rho-dependent termination component of the t'J cluster becomes somewhat leaky, with the readthrough increasing by about twofold. The t'J terminators appear to be less responsive to nutR- and N-mediated antitermination (efficiency of 84-87%) than tL3, which responds to the same antitermination function with an efficiency of 97%. The relationship between the t'J cluster and the same antitermination function with an efficiency of 97%. The relationship between the t'J cluster and the highly N-unresponsive leftward termination signal tJ, which is also located within the J gene region (Gottesman et al., J. Mol. Biol. 140 (1980) 57-75; Honigman, Gene 13 (1981) 299-309), is unknown.

Bacteriophage lambda↗
PubMed2006

Neuromuscular therapeutics by RNA-targeted suppression of ACHE gene expression.

RNA-targeted therapeutics offers inherent advantages over small molecule drugs wherever one out of several splice variant enzymes should be inhibited. Here, we report the use of Monarsen, a 20-mer acetylcholinesterase-targeted antisense agent with three 3'-2'o-methyl-protected nucleotides, for selectively attenuating the stress-induced accumulation of the normally rare, soluble "readthrough" acetylcholinesterase variant AChE-R. Acetylcholine hydrolysis by AChE-R may cause muscle fatigue and moreover, limit the cholinergic anti-inflammatory blockade, yielding inflammation-associated pathology. Specific AChE-R targeting by Monarsen was achieved in cultured cells, experimental animals, and patient volunteers. In rats with experimental autoimmune myasthenia gravis, oral delivery of Monarsen improved muscle action potential in a lower dose regimen (nanomolar versus micromolar), rapid and prolonged manner (up to 72 h versus 2-4 h) as compared with the currently used small molecule anticholinesterases. In central nervous system neurons of both rats and cynomolgus monkeys, systematic Monarsen treatment further suppressed the levels of the proinflammatory cytokines interleukin-1 (IL-1) and IL-6. Toxicology testing and ongoing clinical trials support the notion that Monarsen treatment would offer considerable advantages over conventional cholinesterase inhibitors with respect to dosing, specificity, side effects profile, and duration of efficacy, while raising some open questions regarding its detailed mechanism of action.

Acetylcholine↗
PubMed1999

Molecular characterization of a new furovirus mainly infecting rye.

The complete or almost complete nucleotide sequences were determined for the two RNAs of three different sources of a new furovirus which mainly infects rye and for which the name Soil-borne rye mosaic furovirus (SBRMV) is proposed. The genome organization of this virus is virtually identical to that of Soil-borne wheat mosaic furovirus (SBWMV). However, SBRMV and SBWMV differ considerably in the nucleotide sequences of their genomes and in the derived amino acid sequences of their putative gene products. The sequences of the three sources of SBRMV (two from rye and one from wheat) differ between each other in various parts of their genomes by 1% to 10%. Larger differences were found in the readthrough part of the coat protein readthrough protein. There are no indications that the relationship of the C source from wheat to the other two sources from rye is more distant than the relationship between the two sources from rye. The 3'UTRs of both SBRMV RNAs are remarkable in having a predicted upstream pseudoknot domain (UPD) with seven pseudoknots. The same number was also predicted for the UPD of SBWMV RNA 1 whereas the much shorter UDP of SBWMV RNA 2 may form only four pseudoknots.

3' Untranslated Regions↗
PubMed1985

Identification of a sequence element on the 3' side of AAUAAA which is necessary for simian virus 40 late mRNA 3'-end processing.

Our previous studies of the 3'-end processing of simian virus 40 late mRNAs indicated the existence of an essential element (or elements) downstream of the AAUAAA signal. We report here the use of transient expression analysis to study a functional element which we located within the sequence AGGUUUUUU, beginning 59 nucleotides downstream of the recognized signal AAUAAA. Deletion of this element resulted in (i) at least a 75% drop in 3'-end processing at the normal site and (ii) appearance of readthrough transcripts with alternate 3' ends. Some flexibility in the downstream position of this element relative to the AAUAAA was noted by deletion analysis. Using computer sequence comparison, we located homologous regions within downstream sequences of other genes, suggesting a generalized sequence element. In addition, specific complementarity is noted between the downstream element and U4 RNA. The possibility that this complementarity could participate in 3'-end site selection is discussed.

Base Sequence↗
PubMed1997

Overexpression of human release factor 1 alone has an antisuppressor effect in human cells.

Two eukaryotic proteins involved in translation termination have recently been characterized in in vitro experiments. Eukaryotic release factor 1 (eRF1) catalyzes the release of the polypeptide chain without any stop codon specificity. The GTP-binding protein eRF3 confers GTP dependence to the termination process and stimulates eRF1 activity. We used tRNA-mediated nonsense suppression at different stop codons in a cat reporter gene to analyze the polypeptide chain release factor activities of the human eRF1 and eRF3 proteins overexpressed in human cells. In a chloramphenicol acetyltransferase assay, we measured the competition between the suppressor tRNA and the human release factors when a stop codon was present in the ribosomal A site. Whatever the stop codon (UAA, UAG, or UGA) present in the cat open reading frame, the overexpression of human eRF1 alone markedly decreased translational readthrough by suppressor tRNA. Thus, like the procaryotic release factors RF1 and RF2 in Escherichia coli, eRF1 seems to have an intrinsic antisuppressor activity in human cells. Levels of antisuppression of overexpression of both eRF3 and eRF1 were almost the same as those of overexpression of eRF1 alone, suggesting that eRF1-eRF3 complex-mediated termination may be controlled by the expression level of eRF1. Surprisingly, when overexpressed alone, eRF3 had an inhibitory effect on cat gene expression. The results of cat mRNA stability studies suggest that eRF3 inhibits gene expression at the transcriptional level. This indicates that in vivo, eRF3 may perform other functions, including the stimulation of eRF1 activity.

Blotting, Northern↗
PubMed1983

Molecular cloning of the Pseudomonas carboxypeptidase G2 gene and its expression in Escherichia coli and Pseudomonas putida.

The gene coding for carboxypeptidase G2 was cloned from Pseudomonas sp. strain RS-16 into Escherichia coli W5445 by inserting Sau3A-generated DNA fragments into the BamHI site of pBR322. The plasmid isolated, pNM1, was restriction mapped, and the position of the gene on the 5.8-megadalton insert was pinpointed by subcloning. The expression of carboxypeptidase in E. coli was 100-fold lower than in the Pseudomonas sp. strain. When the cloned gene was subcloned into the Pseudomonas vector pKT230 and introduced into Pseudomonas putida 2440, a 30-fold increase in expression over that obtained in E. coli was observed. High expression (up to 5% soluble protein) was obtained in E. coli by subcloning a 3.1-megadalton Bg/II fragment into the BamHI site of pAT153. The increased expression was orientation dependent and is presumed to be due to transcriptional readthrough from the Tc promoter of the vector. Production of carboxypeptidase was shown to be induced (two-fold) by the presence of folic acid, and the mature protein was shown to be located in the periplasmic space of E. coli.

Carboxypeptidases↗
PubMed1996

Expression of the barley stripe mosaic virus RNA beta "triple gene block".

Genomic RNA beta of barley strip mosaic virus (BSMV) contains four defined open reading frames (ORFs). These include the coat protein (beta a) and a "triple gene block" consisting of the beta b, beta c, and beta d ORFs that overlap one another. Two subgenomic beta RNAs (sgRNA beta 1 and sgRNA beta 2) with sizes of 2.5 and 0.96 kb were identified in BSMV-infected protoplasts, and their transcription initiation sites were mapped to nucleotides 789 and 2327, respectively, of RNA beta by primer extension experiments. In a cell-free wheat germ translation system, genomic RNA beta served as a mRNA only for the 22-kDa coat protein, and sgRNA beta 1 directed synthesis of only the 58-kDA beta b protein. However, with sgRNA beta 2, three proteins with sizes of 14, 17, and 23 kDa were synthesized. Both the 14- and the 23-kDa proteins were recognized by the beta d antibodies in vitro and in vivo. These results demonstrated that the 14-kDa protein was encoded by the beta d ORF and suggested that the 23-kDa protein, designated beta d', is a readthrough product of the amber stop codon of the beta d ORF. Mutagenesis of sgRNA beta 2 revealed that the 17-kDa protein was a product of the beta c ORF. Expression of sgRNA beta 1 and sgRNA beta 2 was also investigated with the chloramphenicol acetyl transferase (CAT) reporter gene in protoplasts coinfected with RNAs alpha and gamma plus chimeric RNA beta derivatives containing the CAT gene in-frame with the beta b, beta c, beta d, or beta d' ORFs. Elimination of the sgRNA beta 1 promoter abolished CAT expression from the beta b-CAT chimeric RNA, and removal of the sgRNA beta 2 promoter prevented CAT expression from the beta c-CAT, beta d-CAT, and beta d'-CAT chimeric RNAs. Taken together, these results demonstrate that the BSMV coat protein is the sole translation product of the genomic RNA beta, whereas sgRNA beta 1 serves as a messenger for translation of the beta b protein, and sgRNA beta 2 functions as a messenger for translation of beta c and beta d and the newly discovered beta d' protein. Additional mutagenesis experiments indicate that beta c is translated by a leaky scanning mechanism.

Base Sequence↗