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PubMed1985

Effects of alterations in the 3' flanking sequence on in vivo and in vitro expression of the yeast SUP4-o tRNATyr gene.

The SUP4-o gene of Saccharomyces cerevisiae codes for an altered tRNATyr capable of suppressing ochre mutations. We constructed mutant SUP4-o genes with deletions in the 3'-flanking sequence and tested each for its ability to suppress ochre mutations in transformed yeast cells. The effects of the different 3' deletions on various aspects of in vitro transcription and RNA processing were also determined, using a yeast cell-free extract. Deletions that leave five or fewer consecutive T residues in the 3'-flanking sequence of SUP4-o were found to result in decreased efficiency of transcription termination, both in vitro and in vivo. Unexpectedly, the suppression strength of each mutant SUP4-o gene is highly correlated with the relative extent of transcription termination at the 3' end of the gene. This result indicates that SUP4-o readthrough transcripts are not efficiently processed to functional suppressor tRNA in yeast cells. Deletions that extend into the T cluster in the 3'-flanking sequence also significantly decrease the ability of SUP4-o to compete for a transcription factor that is limiting in our extracts. This latter finding implies that the 3'-flanking sequence of SUP4 plays a role in transcription factor binding.

Chromosome Deletion↗

PubMed2004

Premature stop codons involved in muscular dystrophies show a broad spectrum of readthrough efficiencies in response to gentamicin treatment.

The suppression levels induced by gentamicin on premature stop codons, caused by primary nonsense mutations found in muscular dystrophy patients, were assessed using a very sensitive dual reporter gene assay. Results show that: (i) the effect of gentamicin on readthrough is similar in cultured cells and in vivo in murine skeletal muscle; (ii) a wide variability of readthrough efficiency is obtained, depending on the mutation tested; (iii) due to the complexity of readthrough regulation, efficiency cannot be predicted by the nucleotide context of the stop codon; (iv) only a minority of premature stop codons found in patients show a significant level of readthrough, and would thus be amenable to this pharmacological treatment, given our present understanding of the problem. These results probably provide an explanation for the relative failure of clinical trials reported to date using gentamicin to treat diseases due to premature stop codons, and emphasize that preliminary assays in cell culture provide valuable information concerning the potential efficiency of pharmacological treatments.

3T3 Cells↗

PubMed1988

Molecular hypotheses for position-effect variegation: anti-sense transcription and promoter occlusion.

There is currently no comprehensive molecular hypothesis to account for position-effect variegation, the mosaic expression of a gene lying near a breakpoint of a chromosomal rearrangement. Here it is proposed that position-effect variegation arises from either anti-sense transcription or from promoter occlusion (transcription readthrough), the former mechanism operating for breakpoints on the 3' side of the affected gene and the latter for breakpoints on the 5' side. Anti-sense transcription will occur in rearrangements that place the anti-sense strand of genes next to a promoter. This anti-sense RNA hybridizes to, and thereby inactivates, sense mRNA transcripts (as anti-sense RNA is known to do). Promoter occlusion may occur in rearrangements that place the affected gene near an open upstream promoter. This promoter drives readthrough transcription that inhibits most normal transcripts. Occasional normal transcripts lead to phenotypic variegation. These hypotheses have three strengths: (i) they predict the major observed features of position-effect variegation including variegated phenotype, stable inheritance, the involvement of rearrangements, only some rearrangements causing variegation, the occurrence of both dominant and recessive variegation, the spreading effect of variegation to several loci, and the conditions required for expression of variegation; (ii) they can plausibly account for features of position-effect variegation that they do not specifically predict; (iii) they lead to a series of novel and testable predictions, including the presence of altered transcripts in rearrangements including position-effect variegation, the location of breakpoints required to cause variegation, and a correlation between the extent of the spreading effect and the length of the novel transcript. These mechanisms can account for several other cases of variegation in addition to classic position-effect variegation. Actual or putative examples of phenotypic variegation due to these mechanisms are known.

Animals↗

PubMed1994

tRNA-directed transcription antitermination.

At least 18 aminoacyl-tRNA synthetase and amino acid biosynthesis genes in several Gram-positive genera appear to be regulated by a common transcription antitermination mechanism. Each gene is induced by limitation for the appropriate amino acid, and not by general amino acid limitation. The mRNA leader regions of these genes exhibit extensive structural conservation. Characterization of the Bacillus subtilis tyrS gene revealed that uncharged tyrosyl-tRNA promotes readthrough of a leader-region terminator; a conformational switch in the leader mRNA between a terminator structure and an antiterminator structure is postulated to mediate antitermination. Two sites of interaction between the tRNA and the leader have been identified by genetic analysis: the tRNA anticodon interacts with a single codon displayed at a precise position in the leader-region structure, and the acceptor end of the tRNA interacts with a side-bulge on the antiterminator.

Amino Acids↗

PubMed1994

Characterization and expression of a gene encoding serine tRNA5 from Escherichia coli.

The genes for translational components frequently are located together on the Escherichia coli genome. We have reported previously that the gene for a serine tRNA lies directly downstream from infA, the gene encoding initiation factor IF1. Here we characterize this tRNA gene, named serW. The serW gene expresses a minor form of serine tRNA(GGA) which recognizes the most frequently used serine codons, UCC and UCU. Two promoters were identified by S1 nuclease mapping: P1, which lies about 72 bp upstream from the structural gene; and P2, which lies about 35 bp upstream. Expression from P1 and P2 is comparable under conditions of rapid growth. The P2 promoter is followed by a GC-rich element characteristic of promoters regulated by ppGpp. A putative hairpin structure followed by a stretch of U residues about 25 nucleotides following the mature tRNA sequence resembles a rho-independent termination signal. The upstream gene, infA, is followed by a transcriptional terminator, but S1 mapping shows considerable readthrough. This serW expression appears to rely both on its own promoters and on promoters further upstream. The downstream gene, encoding an unidentified protein of about 100 kDa, is expressed in the opposite orientation and also is followed by a termination signal. Therefore serW is expressed both as a monocistronic gene and in combination with infA.

Base Sequence↗

PubMed1995

[PSI] and [URE3] as yeast prions.

[URE3] is a non-Mendelian genetic element that mimics recessive mutations in the chromosomal URE2 gene making cells derepressed for nitrogen catabolic enzymes. [PSI] is a non-Mendelian enhancer of readthrough of translational termination similar in its effects to some mutations in the chromosomal SUP35 gene. Three lines of evidence led to the proposal that both [URE3] and [PSI] are prions, infectious proteins analogous to the scrapie agent mediating transmissible spongiform encephalopathies of mammals. 1) Both [PSI] and [URE3] are reversibly curable. 2) [PSI] propagation requires SUP35 and [URE3] propagation requires URE2 with recessive chromosomal mutants having the same phenotypes as the presence of the respective dominant non-Mendelian element. 3) Overproduction of Sup35p and Ure2p increases the frequency of cells acquiring [PSI] or [URE3], respectively.

Amino Acid Sequence↗

PubMed2004

Monitoring uncharged tRNA during transcription of the Bacillus subtilis glyQS gene.

Expression of the Bacillus subtilis glyQS gene, encoding glycyl-tRNA synthetase, depends on stabilization of an antiterminator element during transcription of the 5' region of the mRNA by binding of uncharged tRNA(Gly). The glyQS gene is a member of the T box family of genes, all of which are involved in generation of charged tRNA. Each gene in this family exhibits an increase in readthrough of a termination signal located upstream of the start of the coding sequence in response to a decrease in the ratio of charged to uncharged tRNA. Many structural features of T box RNAs that are necessary for tRNA-dependent antitermination have been defined, but little is known about the timing or sequence of events that lead to a productive interaction with uncharged tRNA and discrimination against charged tRNA. To investigate these issues, transcription complexes were blocked artificially at specific positions along the leader sequence and tested for the ability to recognize tRNA. Although the sequence element that binds the tRNA anticodon is located more than 100 nt before the termination signal, complexes with nascent transcripts extending to just upstream of the termination site were still competent for antitermination. This result indicates that the transcript can fold into a receptive structure in the absence of the tRNA, and that tRNA is not necessary prior to this point. A mimic of charged tRNA(Gly) inhibited antitermination by uncharged tRNA unless the leader RNA-tRNA(Gly) complexes contained the complete antiterminator. These results suggest that the transcription complex can interact with either uncharged or charged tRNA until it approaches the termination point, allowing maximal flexibility in monitoring the ratio of charged to uncharged tRNA.

Bacillus subtilis↗

PubMed1994

Characterization of the gene encoding the human transcriptional elongation factor TFIIS.

The transcriptional elongation factor TFIIS causes stimulation of RNA polymerase II elongation and readthrough of some of the elongation blocks. We present cloning and sequence characterization of the human TFIIS gene and a pseudogene. The intron-less organization of both of these genes indicates that previously identified cDNAs which suggested the presence of an intron were the products of cloning artifacts. The gene is organized in an uninterrupted ORF which codes for 301 amino acids, whereas the pseudogene lacks an ORF able to code for a full-length protein. The potential promoter for the gene has two putative GC-box-type consensus sequences, two CCAAT-box consensus sequences, and is bounded by a human Alu sequence. Two potential transcriptional termination signal sequences downstream from the consensus polyadenylation signal are proposed.

Base Sequence↗

PubMed1991

Salmonella typhimurium prfA mutants defective in release factor 1.

Mutations have been characterized that map in the prfA gene of Salmonella typhimurium. These weak amber suppressors show increased readthrough of UAG but not UAA or UGA codons. Some hemA mutants exhibit a similar suppressor activity due to transcriptional polarity on prfA. All of the suppressors mapping in prfA are recessive to the wild type. Two mutant prfA genes were cloned onto plasmids, and their DNA sequences were determined. A method was devised for transferring the sequenced mutant alleles back to their original location in S. typhimurium via an Escherichia coli recD strain that carries the entire S. typhimurium hemA-prfA operon as a chromosomal insertion in trp. This reconstruction experiment showed that the mutations sequenced are sufficient to confer the suppressor phenotype.

Cloning, Molecular↗

PubMed1995

Nucleotide sequence of beet mild yellowing virus RNA.

The complete nucleotide sequence of the genomic RNA of beet mild yellowing virus, isolate 2ITB, is reported. The RNA consists of 5722 nucleotides and contains six long open reading frames which conform to the arrangement characteristic of Subgroup 2 luteoviruses. The three 3'-proximal open reading frames, which encode the viral coat protein, a putative movement protein and the Readthrough Domain, are highly homologous to the corresponding genes of beet western yellows luteovirus while the three 5'-proximal open reading frames are more closely related to the corresponding genes of cucurbit aphid borne yellows luteovirus. The sequence data thus indicate that beet mild yellowing virus should be considered a distinct virus rather than a strain of beet western yellows virus.

Amino Acid Sequence↗

PubMed1998

The genome organization of the broad bean necrosis virus (BBNV).

The genome of the broad bean necrosis virus Oita-isolate (BBNV-O) [RNA1 (6.0 kb), RNA2 (2.8 kb) and RNA3 (2.4 kb)] was cloned and sequenced. Computer analysis indicates that methyltransferase, helicase and RNA-dependent RNA polymerase (RdRp) motifs are present in RNA1. The viral capsid protein (CP) cistron is located at the 5' terminal end of RNA2 and the Mr of CP (20 K) is close to that determined by SDS-PAGE analysis. An ochre codon (UAA) in the CP cistron is thought to be partially suppressed to produce a large readthrough protein. RNA3 possesses typical motifs of triple gene block proteins, which are also reported in several other plant viruses. The furovirus genome organization and phylogenetic analysis using RdRp and CP amino acid sequences suggest that BBNV is closely related to potato mop-top virus (PMTV), but is relatively distantly related to other furoviruses. The data also suggest that the genus Furovirus should be separated into several genera: the prototypical genus Furovirus, which excludes the following viruses: the PMTV group including BBNV; the beet necrotic yellow vein virus (BNYVV) group; and the peanut clump virus (PCV) group.

Amino Acid Sequence↗

PubMed2005

Rsp5 ubiquitin ligase modulates translation accuracy in yeast Saccharomyces cerevisiae.

Rsp5p is an essential yeast ubiquitin protein ligase that ubiquitinates multiple proteins involved in various processes. Recent studies indicate that ubiquitination also affects translation. Here, we show that the strain with the rsp5-13 mutation exhibits altered sensitivity to antibiotics and a slower rate of translation. Using a sensitive dual-gene reporter system, we demonstrate that stop codon readthrough efficiency is decreased in the rsp5-13 mutant, while both +1 and -1 frameshifting were unaffected. The effect of the rsp5-13 mutation on readthrough could be reversed by increased expression of ubiquitin and partially suppressed by overproduction of the elongation factor eEF1A. As assessed by fluorescence in situ hybridization, the rsp5-13 mutant cells accumulate tRNA nuclear pools, perhaps depleting tRNA from the cytoplasm. Nuclear accumulation of tRNA is observed only when rsp5-13 cells are grown in media with high amino acid content. This defect, also reversed by overproduction of the elongation factor eEF1A, may be the primary reason for altered translational decoding accuracy.

Antifungal Agents↗

PubMed1982

Bioassay for components regulating eukaryotic gene expression: a chromosomal factor involved in the generation of histone mRNA 3' termini.

We have adapted the oocyte injection procedure for the detection of regulatory components involved in the transcription of a eukaryotic mRNA gene. Injection of the histone gene repeat h22 DNA of Psammechinus miliaris into the Xenopus oocyte nucleus results in correct initiation of the histone mRNAs, but readthrough by RNA polymerase occurs at the 3' end of the H3 histone gene (Hentschel, C. C., Probst, E. & Birnstiel, M. L. (1980) Nature (London) 288, 100-102). Coinjection into the oocyte of a chromosomal salt wash fraction derived from sea urchin embryos results in the generation of authentic 3' termini of the histone H3 mRNA. We have partially purified the protein component by column chromatography and density gradient centrifugation. The regulatory factor binds to heparin columns and, hence, has the properties anticipated of an RNA- or DNA-binding protein. The sedimentation coefficient of the active component was determined to be about 12 S, suggesting a molecular weight of 200,000-250,000.

Animals↗

PubMed2004

Molecular characterization of NADase-streptolysin O operon of hemolytic streptococci.

Whether slo, the gene encoding streptolysin O (SLO), a streptococcal cytolysin, has its own promoter or not is unsettled as yet. Present analyses demonstrate that slo is a member of an operon covering the upper-stream nusG and nga (NADase) genes, from which transcription of slo proceeds polycistronically, and major transcript is produced by readthrough from nga promoter. Mutational conversion of the sixth nucleotide T at the putative -10 region of chromosomal nga gene into C caused a drastic decrease in both NADase and SLO activities and the disappearance of the two corresponding mRNA bands from the Northern blot profile. The initiation site of the transcription was determined at 56 bp upstream (NusG gene) and 25 bp upstream (NADase gene) of each initiation codon. Although the promoter region of slo gene is highly conserved between group A and C streptococci, the proper slo promoter is nonfunctional in group C strain H46A. Moreover, commonly conserved arrangement was limited to the nusG-nga-orf1-slo region. These results indicate an intimate relationship between NADase and SLO in the regulation of their biosynthesis. Additional results suggest that NADase, synthesized as precursor with feeble activity, is activated by removing the carboxyl terminal region during or after secretion into culture medium.

3' Flanking Region↗

PubMed1983

Measurement of suppressor transfer RNA activity.

Transfer RNA (tRNA) suppression of nonsense mutations in prokaryotic systems has been widely used to study the structure and function of different prokaryotic genes. Through genetic engineering techniques, it is now possible to introduce suppressor (Su+) tRNA molecules into mammalian cells. A quantitative assay of the suppressor tRNA activity in these mammalian cells is described; it is based on the amount of tRNA-mediated readthrough of a terminating codon in the influenza virus NS1 gene after the cells are infected with virus. Suppressor activity in L cells continuously expressing Su+ (tRNAtyr) was 3.5 percent and that in CV-1 cells infected with an SV40- Su+ (tRNAtyr) recombinant was 22.5 percent.

Animals↗

PubMed1991

UAG readthrough is not increased in vivo by Moloney murine leukemia virus infection.

Expression of the pol gene of the murine leukemia viruses is subject to translational control at the UAG termination codon of the upstream gene gag. Previous experiments have suggested that: i) Moloney murine leukemia virus infection induces a tRNA(Gln)iii) in an in vitro system using the tobacco mosaic virus as template, this tRNA is able to increase readthrough at the UAG codon [1]. Here we demonstrate that, in vivo, Moloney murine leukemia virus infection does not increase translational readthrough at either the tobacco mosaic virus or the Moloney murine leukemia virus UAG stop codons.

3T3 Cells↗

PubMed1990

Readthrough transcription occurs at the rho dependent signal F1 TIV in suppressor cells.

Suppressor cells infected with bacteriophage f1 yield phage encoded gene IV transcripts longer than those present in the supo host and identical to those found in a rho- host. However, such longer transcripts do not appear in the suppressor-infected cell when, by changing the translation frame of gene IV, the ribosome is not allowed to proceed to the end of the gene IV message and thus to reach the rho dependent transcription terminator f1 TIV. This suggests that ribosome movement beyond the natural gene IV stop codon disturbs the activity of that termination signal. In contrast to the rho- behaviour, the suppressor does not accumulate high levels of gene IV messages indicating that the accumulation occurring in the rho- mutant may not be a primary effect of the readthrough per se.

Base Sequence↗

PubMed2006

Regulated readthrough: a new method for the alternative tagging and targeting of recombinant proteins.

We report here a new method for the alternative peptide tagging of recombinant proteins from mammalian cell lines. This method, which we called regulated readthrough, exploits the property of aminoglycoside antibiotics to promote translational readthrough of nonsense codons. The basic expression cassette includes a translational fusion between a gene of interest and a membrane targeting peptide, which are separated by a nonsense codon. In the presence of an aminoglycoside antibiotic, translational readthrough is promoted and results in the targeting of the fusion protein to the cell membrane, thus allowing the efficient flow cytometry-based isolation of cells expressing very high levels of recombinant protein. For downstream applications requiring the production of soluble recombinant protein, the cells are cultured in the absence of aminoglycoside, leading to an efficient translational termination. By combining different translation termination signals that exhibit various susceptibilities to aminoglycoside-mediated translational readthrough with flow cytometry capabilities, it is possible to use this technology for other applications such as functional library screening or monitoring the stability of recombinant protein production.

Amino Acid Sequence↗