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PubMed1991

The product of the yeast UPF1 gene is required for rapid turnover of mRNAs containing a premature translational termination codon.

mRNA decay rates often increase when translation is terminated prematurely due to a frameshift or nonsense mutation. We have identified a yeast gene, UPF1, that codes for a trans-acting factor whose function is necessary for enhanced turnover of mRNAs containing a premature stop codon. In the absence of UPF1 function, frameshift or nonsense mutations in the HIS4 or LEU2 genes that normally cause rapid mRNA decay fail to have this effect. Instead, the mRNAs decay at rates similar to the corresponding wild-type mRNAs. The stabilization of frameshift or nonsense mRNAs observed in upf1- strains does not appear to result from enhanced readthrough of the termination signal. Loss of UPF1 function has no effect on the accumulation or stability of HIS4+ or LEU2+ mRNA, suggesting that the UPF1 product functions only in response to a premature termination signal. When we examined the accumulation and stability of other wild-type mRNAs in the presence or absence of UPF1, including MAT alpha 1, STE3, ACT1, PGK1, PAB1, and URA3 mRNAs, only the URA3 transcript was affected. On the basis of these and other results, the UPF1 product appears to participate in a previously uncharacterized pathway leading to the degradation of a limited class of yeast transcripts.

Base Sequence↗

PubMed1996

What role might lampbrush chromosomes play in maternal gene expression?

The biological significance of lampbrush chromosomes from urodelan amphibians is far from being elucidated. Their particularly well developed lateral loops are the site of intense transcriptional activity, which can be visualized in electron microscopy using the Miller spreading procedure. All transcription units functioning in lampbrush loops synthesize RNA at a maximum rate. In situ hybridization has provided evidence for transcription of both unique coding sequences and highly repetitive sequences. The role of lampbrush transcripts in the production of maternal information remains unclear. RNAs transcribed from unique coding sequences are exported to the cytoplasm; there, they contribute either to maintaining the required level of maternal messenger RNA in a basal state during late oogenesis, or to increasing the store of these maternal RNAs throughout oocyte growth, i.e., until stage VI. For repetitive sequences, their intense transcription appears to be non-productive, in that RNAs are not translatable and might be useless products of readthrough transcription. The non-productive transcription of repetitive sequences, the expression of which is directly related to hyperdevelopment of lateral loops, raises the issue of the role of lampbrush chromosome transcription.

Animals↗

PubMed1987

Nucleotide sequence analysis of Tn4551: use of ermFS operon fusions to detect promoter activity in Bacteroides fragilis.

The Bacteroides pBI136 clindamycin resistance (Ccr) determinant from the composite transposon Tn4551 was cloned onto the shuttle plasmid pFD160, and the regions necessary for expression in Bacteroides fragilis were determined. These results suggested that transcriptional regulatory signals required for Ccr were located in the Tn4551 direct repeat sequence (DRS) adjacent to the resistance determinant. Analysis of the nucleotide sequence of this region revealed that the Ccr structural gene, 798 base pairs (bp), was located 17 bp from the terminus of the DRS and that this gene (ermFS) differed from ermF (pBF4) by one amino acid. The DRS element was found to be 1,155 bp and appeared to contain the ermFS transcription start signals. The DRS structure was typical of insertion sequence elements isolated from other bacterial species, and its termini were characterized by 25-bp regions of imperfect dyad symmetry. The DRS was dominated by a 978-bp open reading frame, which terminated in the left inverted repeat 27 bp from the ermFS start codon, and weak amino acid sequence homology was observed with the putative transposase of IS3. Promoter activity of the DRS in B. fragilis was demonstrated by in vitro construction of operon fusions with a promoterless ermFS gene followed by transformation of the recombinant plasmids with selection for resistance to clindamycin. The location of one DRS promoter was identified by using the ermFS fusions and then verified by in vitro mutagenesis of the site with single-stranded linkers. Northern blot (RNA blot) analysis of total RNA from B. fragilis strains containing pBI136 or ermFS recombinant plasmids confirmed the location of this promoter and indicated that it was used in vivo by Tn4551. A second DRS promoter, which activated ermFS transcription by readthrough of the large DRS open reading frame, was also identified by the Northern blot analysis. The bicistronic ermFS message was not observed in strains containing a complete copy of Tn4551, and the possibility of transcriptional regulation is discussed.

Amino Acid Sequence↗

PubMed1988

On the specificity and effects on transcription of P-element insertions at the yellow locus of Drosophila melanogaster.

Fourteen P-element insertion mutants at the yellow (y) gene of D. melanogaster have been analysed by restriction mapping and DNA sequencing. The distribution of the insertion sites is nonrandom, with a preferred target site in the 5'-transcribed but non-translated leader of the gene. Transcription analysis indicates that P-elements inserted into the transcribed region of y can terminate y transcription. While the great majority of the P-element insertions into the preferred target site abolish y activity, this loss of activity is not due to disruption of the insertion site since imprecise excision events which do not restore the wild type sequence at the insertion site can restore y function. We further demonstrate that a mutant caused by a 221bp P-element insertion into the 5' transcribed but non-translated region, which show partial y activity, produces a larger mutant transcript consistent with it being a product of transcriptional readthrough. We present results which suggest that imprecise excision events which restore (or partially restore) y activity can occur by deleting P-element internal sequences which normally act to terminate y transcription.

Animals↗

PubMed2005

The polerovirus minor capsid protein determines vector specificity and intestinal tropism in the aphid.

Aphid transmission of poleroviruses is highly specific, but the viral determinants governing this specificity are unknown. We used a gene exchange strategy between two poleroviruses with different vectors, Beet western yellows virus (BWYV) and Cucurbit aphid-borne yellows virus (CABYV), to analyze the role of the major and minor capsid proteins in vector specificity. Virus recombinants obtained by exchanging the sequence of the readthrough domain (RTD) between the two viruses replicated in plant protoplasts and in whole plants. The hybrid readthrough protein of chimeric viruses was incorporated into virions. Aphid transmission experiments using infected plants or purified virions revealed that vector specificity is driven by the nature of the RTD. BWYV and CABYV have specific intestinal sites in the vectors for endocytosis: the midgut for BWYV and both midgut and hindgut for CABYV. Localization of hybrid virions in aphids by transmission electron microscopy revealed that gut tropism is also determined by the viral origin of the RTD.

Animals↗

PubMed2001

[Function of the leaky UGA codon in ssRNA plant virus genomes].

The opal mutation (UGA) at nucleotide 2670-2672 in the replicase gene of the attenuated tomato mosaic virus K (ToMV-K) mainly contributes to the virus attenuation based on a series of studies on the viral attenuation mechanism. From analysis and comparison between the replicase gene mutation point of ToMV-K and the related regions of all plant viruses containing the leaky UGA, we have found that some characters, including the CGG motif, alpha-helix structure and some specific amino acids, are, presumably, able to helpfully confer the readthrough mechanism. Finally, some other ssRNA plant viruses like PVX, PVY, CMV have been analyzed. We found that their genomic modifications and viral attenuations could be explored according to the mutation mode of the ToMV-K replicase.

Amino Acid Sequence↗

PubMed1998

A molecular switch controlling competence and motility: competence regulatory factors ComS, MecA, and ComK control sigmaD-dependent gene expression in Bacillus subtilis.

Bacillus subtilis, like many bacteria, will choose among several response pathways when encountering a stressful environment. Among the processes activated under growth-restricting conditions are sporulation, establishment of motility, and competence development. Recent reports implicate ComK and MecA-ClpC as part of a system that regulates both motility and competence development. MecA, while negatively controlling competence by inhibiting ComK, stimulates sigmaD-dependent transcription of genes that function in motility and autolysin production. Both ComK-dependent and -independent pathways have been proposed for MecA's role in the regulation of motility. Mutations in mecA reduce the transcription of hag. encoding flagellin, and are partially suppressed by comK in both medium promoting motility and medium promoting competence. Reduced sigmaD levels are observed in mecA mutants grown in competence medium, but no change in sigmaD concentration is detected in a comK mutant. The comF operon, transcription of which requires ComK, is located immediately upstream of the operon that contains the flgM gene, encoding the sigmaD-specific antisigma factor. An insertion mutation that disrupts the putative comF-flgM transcription unit confers a phenotype identical to that of the comK mutant with respect to hag-lacZ expression. Expression of a flgM-lacZ operon fusion is reduced in both sigD and comK mutant cells but is abolished in the sigD comK double mutant. Reverse transcription-PCR examination of the comF-flgM transcript indicates that readthrough from comF into the flgM operon is dependent on ComK. ComK negatively controls the transcription of hag by stimulating the transcription of comF-flgM, thereby increasing the production of the FlgM antisigma factor that inhibits sigmaD activity. There likely exists another comK-independent mechanism of hag transcription that requires mecA and possibly affects the sigmaD concentration in cells undergoing competence development.

Bacillus subtilis↗

PubMed2006

Transcription elongation factor S-II is required for definitive hematopoiesis.

Transcription elongation factor S-II/TFIIS promotes readthrough of transcriptional blocks by stimulating nascent RNA cleavage activity of RNA polymerase II in vitro. The biologic significance of S-II function in higher eukaryotes, however, remains unclear. To determine its role in mammalian development, we generated S-II-deficient mice through targeted gene disruption. Homozygous null mutants died at midgestation with marked pallor, suggesting severe anemia. S-II(-/-) embryos had a decreased number of definitive erythrocytes in the peripheral blood and disturbed erythroblast differentiation in fetal liver. There was a dramatic increase in apoptotic cells in S-II(-/-) fetal liver, which was consistent with a reduction in Bcl-x(L) gene expression. The presence of phenotypically defined hematopoietic stem cells and in vitro colony-forming hematopoietic progenitors in S-II(-/-) fetal liver indicates that S-II is dispensable for the generation and differentiation of hematopoietic stem cells. S-II-deficient fetal liver cells, however, exhibited a loss of long-term repopulating potential when transplanted into lethally irradiated adult mice, indicating that S-II deficiency causes an intrinsic defect in the self-renewal of hematopoietic stem cells. Thus, S-II has critical and nonredundant roles in definitive hematopoiesis.

Animals↗

PubMed1996

Structure of the C-terminal end of the nascent peptide influences translation termination.

The efficiency of translation termination at NNN NNN UGA A stop codon contexts has been determined in Escherichia coli. No general effects are found which can be attributed directly to the mRNA sequences itself. Instead, termination is influenced primarily by the amino acids at the C-terminal end of the nascent peptide, which are specified by the two codons at the 5' side of UGA. For the penultimate amino acid (-2 location), charge and hydrophobicity are important. For the last amino acid (-1 location), alpha-helical, beta-strand and reverse turn propensities are determining factors. The van der Waals volume of the last amino acid can affect the relative efficiency of stop codon readthrough by the wild-type and suppressor forms of tRNA(Trp) (CAA). The influence of the -1 and -2 amino acids is cooperative. Accumulation of an mRNA degradation intermediate indicates mRNA protection by pausing ribosomes at contexts which give inefficient UGA termination. Highly expressed E.coli genes with the UGA A termination signal encode C-terminal amino acids which favour efficient termination. This restriction is not found for poorly expressed genes.

Amino Acid Sequence↗

PubMed1995

Functional comparison of the Mus musculus molossinus and Mus musculus domesticus Sry genes.

The Sry gene functions as a genetic switch initiating testicular development of the indifferent mammalian gonad. The Mus musculus molossinus Sry open reading frame (ORF) encodes a 395-amino acid transcription factor (mSry) that specifically binds and bends DNA through its N-terminal HMG domain and activates transcription through its long C-terminal (residues 144-366) glutamine/histidine-rich activation domain. The M. m. domesticus Sry ORF encodes a highly homologous, truncated protein (dSry) of approximately 230 amino acids, and the molecular basis for truncation is a point mutation that creates an amber stop codon within the activation domain. The mSry protein activates transcription of a Sry-responsive reporter gene in HeLa cells, but dSry does not. Gene swapping and in vitro DNA binding experiments revealed that lack of transcriptional activation by dSry was not the result of polymorphisms within the first 137 amino acids of the protein. Direct analysis of the C-terminal glutamine/histidine-rich domain revealed that dSry lacked a functional transcriptional activation domain. Fusion of the GAL4 DNA-binding domain to the C-terminal deletion mutants of the GAL4-mSry chimeric protein indicated that residues 263-345 of the glutamine/histidine-rich domain were necessary for high level transactivation. Furthermore, readthrough of the premature amber stop codon by transfer RNA suppression resulted in a strong GAL4-dSry transactivator. This demonstrated that the premature stop codon is the only polymorphism responsible for the inability of the dSry glutamine/histidine-rich region to transactivate.

Animals↗

PubMed1997

The efficiency of a cis-cleaving ribozyme in an mRNA coding region is influenced by the translating ribosome in vivo.

A cis -cleaving hammerhead ribozyme (Rz) expression system (3A'-Rz) in Escherichia coli has been constructed that can be used to study the involvement of factors that affect ribozyme cleavage in vivo . The ribozyme sequence is placed in the coding region of 3A' mRNA, which is expressed from a semi-synthetic translation assay gene. The size and the 5'-end sequences of the 3' cleavage fragments were determined and the efficiencies of different Rz variants were measured by quantitative primer extension. It is shown that one of the semi-active constructs (3A'-RzIII) can be used as an indicator for ribosomes that read through or terminate at a stop codon upstream of the Rz hammerhead sequence in the mRNA. Readthrough of the stop codon in an uncleaved mRNA gives a full length 3A' protein. Termination at the stop codon upstream of the ribozyme sequence gives a shortened termination product. However, the mRNA fragment that should arise as a result of the auto-cleavage does not give rise to any detectable corresponding truncated protein. Besides studies on translating ribosomes, the 3A'-Rz system can be used to isolate mutant strains that are changed in ribozyme activity either from internal base alterations, or changed interacting host factors.

Base Sequence↗

PubMed1989

Polyadenylation at correct sites in genome RNA is not required for retrovirus replication or genome encapsidation.

RNA transcripts polyadenylated at sites derived from flanking cellular DNA (readthrough transcripts) make up about 15% of the viral RNA in cells infected with avian leukosis virus. To test the functionality of such transcripts, a virus was created by introducing two mutations into the AAUAAA polyadenylation signal of Rous-associated virus 1, converting it to AAGGAA. The replication of this virus was not greatly affected at any level. However, less than 1% of viral transcripts produced during mutant virus replication were cleaved and polyadenylated at the correct site within viral long terminal repeat-related sequence. These results imply that readthrough transcripts, which are produced during normal viral replication, are polyadenylated and packaged into virions as normal transcripts and can serve as RNA genomes in the next round of replication. These results show that polyadenylation within virus-related sequences is not a necessary requirement for virus replication and that readthrough transcripts have the necessary properties to be intermediates in the process of transduction of cellular sequences.

Animals↗

PubMed1995

A mutant allele of the SUP45 (SAL4) gene of Saccharomyces cerevisiae shows temperature-dependent allosuppressor and omnipotent suppressor phenotypes.

Using a plasmid-based termination-read-through assay, the sal4-2 conditional-lethal (temperature-sensitive) allele of the SUP45 (SAL4) gene was shown to enhance the efficiency of the weak ochre suppressor tRNA SUQ5 some 10-fold at 30 degrees C. Additionally, this allele increased the suppressor efficiency of SRM2-2, a weak tRNA(Gln) ochre suppressor, indicating that the allosuppressor phenotype is not SUQ5-specific. A sup+ sal4-2 strain also showed a temperature-dependent omnipotent suppressor phenotype, enhancing readthrough of all three termination codons. Combining the sal4-2 allele with an efficient tRNA nonsense suppressor (SUP4) increased the temperature-sensitivity of that strain, indicating that enhanced nonsense suppressor levels contribute to the conditional-lethality conferred by the sal4-2 allele. However, UGA suppression levels in a sup+ sal4-2 strain following a shift to the non-permissive temperature reached a maximum significantly below that exhibited by a non-temperature sensitive SUP4 suppressor strain. Enhanced nonsense suppression may not therefore be the primary cause of the conditional-lethality of this allele. These data indicate a role for Sup45p in translation termination, and possibly in an additional, as yet unidentified, cellular process.

Alleles↗

PubMed1992

Transcription frequency modulates the efficiency of an attenuator preceding the rpoBC RNA polymerase genes of Escherichia coli: possible autogenous control.

Expression of the rpoBC genes encoding the beta and beta' RNA polymerase subunits of Escherichia coli is autogenously regulated. Although previous studies have demonstrated a post-transcriptional feedback mechanism, complex transcriptional controls of rpoBC expression may also contribute. We show that an attenuator (rpoBa) separating the ribosomal protein (rpl) genes from the rpoBC genes in the rplKAJLrpoBC gene cluster is modulated in its efficiency in response to changes in the frequency of transcription initiated by promoters located upstream. A series of rplJLrpoBalacZ transcriptional fusions was constructed on lambda vectors in which transcription into the rpoBa attenuator was varied by using a variety of promoters with different strengths. beta-galactosidase assays performed on monolysogens of the recombinant phage show that with transcription increasing over a 40-fold range, readthrough of rpoBa decreases from 61% to 19%. In contrast, two other well-characterized terminators show nearly constant efficiencies over a similar range of transcription frequencies. Using a set of phage P22 ant promoter variants with single-nucleotide changes in the promoter consensus sequences also demonstrates that the modulation of rpoBa function appears to be unrelated to the phenomenon of 'factor-independent antitermination' reported by others. The implications for autogenous control of RNA polymerase synthesis are discussed.

Cloning, Molecular↗

PubMed1981

Direct measurement of absolute suppressor efficiency.

We have devised a system for measuring the degree of translational readthrough past a nonsense mutation which is based upon the quantitation of the two translation products, the suppressed polypeptide and the nonsense fragment. The absolute efficiency of four different amber suppressors (Su1, Su2, Su3, and Su7) has been determined at two unique amber sites in the structural gene for the beta subunit of Escherichia coli RNA polymerase.

DNA-Directed RNA Polymerases↗

PubMed2004

The plant exon finder: a tool for precise detection of exons using a T-DNA-based tagging approach.

We have developed a new tool, named the plant exon finder (PEF), for identifying exons in plant genome sequences as an applied technique of T-DNA insertional mutagenesis. The T-DNA constructs contain a heat-shock gene promoter or the cauliflower mosaic virus (CaMV) 35S promoter, followed by the first exon of an Arabidopsis gene with its start codon and the intron donor sequence facing the T-DNA left border (LB) in order to trap exons in the genome. The constructs were used to make transgenic Arabidopsis plants. We generated 280 transgenic lines and identified 156 T-DNA-tagged readthrough transcripts by reverse transcriptase-polymerase chain reaction (RT-PCR) using an oligo(dT)-linker primer and a T-DNA-specific primer. Sequence analysis of the RT-PCR products revealed that 18 of them carried cDNAs processed by the use of an intron acceptor sequence adjacent to T-DNA insertion sites and 11 of them were in-frame fusions. In one case, the readthrough transcript trapped an exon located 1.6 kb downstream of the site of the insertion.

Arabidopsis↗

PubMed1994

Erythroid cell-specific determinants of alpha-globin mRNA stability.

Although globin mRNAs are considered prototypes of highly stable messages, the mechanisms responsible for their longevity remain largely undefined. As an initial step in identifying potential cis-acting elements or structures which contribute to their stability, we analyzed the defect in expression of a naturally occurring alpha 2-globin mutant, alpha Constant Spring (CS). The CS mutation is a single-base change in the translation termination codon (UAA-->CAA) that allows the ribosome to read through into the 3' nontranslated region (NTR). The presence of CS mRNA in transcriptionally active erythroid precursors and its absence (relative to normal alpha-globin mRNA) in the more differentiated transcriptionally silent erythrocytes suggest that this mutation disrupts some feature of the alpha-globin mRNA required for its stability. Using a transient transfection system, we demonstrate that in murine erythroleukemia cells the CS mRNA is unstable compared with the normal alpha 2-globin mRNA. The analyses of several other naturally occurring and site-directed mutant alpha-globin genes in murine erythroleukemia cells indicate that entry of a translating ribosome into the 3' NTR targets the message for accelerated degradation in erythroid cells. In contrast, both the CS and alpha 2-globin mRNAs are stable in several nonerythroid cell lines. These results suggest that translational readthrough disrupts a determinant associated with the alpha 2-globin 3' NTR which is required for mRNA stability in erythroid cells.

Animals↗

PubMed1988

Characterization of the termination phenotypes of rifampicin-resistant mutants.

Rifampicin-resistant (Rifr) mutations map in the rpoB gene encoding the beta subunit of Escherichia coli RNA polymerase. We have examined the effect of each of the 17 sequenced Rifr mutations in our collection on transcription termination. The effect of each Rifr mutation was measured at three types of terminators: simple terminators requiring only RNA polymerase to terminate in vitro, and complex terminators requiring either Rho or Tau for in-vitro termination. Almost every Rifr allele examined (14/17) affected readthrough at one or more of these terminators. We found that mutations with similar termination phenotypes were clustered suggesting functional specialization within the region of rpoB defined by the Rifr mutations. The interaction of the Rifr mutations with the defective rho15 allele was also investigated. Only two Rifr mutations suppress the termination defect of rho15 strains. We discuss models to explain how this region of the beta polypeptide might be involved in the process of transcription termination.

Drug Resistance, Microbial↗