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PubMed2006

Construction of improved temperature-sensitive and mobilizable vectors and their use for constructing mutations in the adhesin-encoding acm gene of poorly transformable clinical Enterococcus faecium strains.

Inactivation by allelic exchange in clinical isolates of the emerging nosocomial pathogen Enterococcus faecium has been hindered by lack of efficient tools, and, in this study, transformation of clinical isolates was found to be particularly problematic. For this reason, a vector for allelic replacement (pTEX5500ts) was constructed that includes (i) the pWV01-based gram-positive repAts replication region, which is known to confer a high degree of temperature intolerance, (ii) Escherichia coli oriR from pUC18, (iii) two extended multiple-cloning sites located upstream and downstream of one of the marker genes for efficient cloning of flanking regions for double-crossover mutagenesis, (iv) transcriptional terminator sites to terminate undesired readthrough, and (v) a synthetic extended promoter region containing the cat gene for allelic exchange and a high-level gentamicin resistance gene, aph(2'')-Id, to distinguish double-crossover recombination, both of which are functional in gram-positive and gram-negative backgrounds. To demonstrate the functionality of this vector, the vector was used to construct an acm (encoding an adhesin to collagen from E. faecium) deletion mutant of a poorly transformable multidrug-resistant E. faecium endocarditis isolate, TX0082. The acm-deleted strain, TX6051 (TX0082Deltaacm), was shown to lack Acm on its surface, which resulted in the abolishment of the collagen adherence phenotype observed in TX0082. A mobilizable derivative (pTEX5501ts) that contains oriT of Tn916 to facilitate conjugative transfer from the transformable E. faecalis strain JH2Sm::Tn916 to E. faecium was also constructed. Using this vector, the acm gene of a nonelectroporable E. faecium wound isolate was successfully interrupted. Thus, pTEX5500ts and its mobilizable derivative demonstrated their roles as important tools by helping to create the first reported allelic replacement in E. faecium; the constructed this acm deletion mutant will be useful for assessing the role of acm in E. faecium pathogenesis using animal models.

Adhesins, Bacterial↗
PubMed2026

Molecular Determinants and Therapeutic Targeting of Stop Codon Readthrough in Eukaryotic Translation.

Accurate translation termination is essential for proteome integrity and in eukaryotes is primarily governed by the release factors eRF1 and eRF3, which ensure precise recognition of stop codons and efficient release of nascent polypeptides. However, proteome integrity is challenged by mutations that generate premature termination codons (PTCs), leading to truncated, nonfunctional proteins and degradation of the aberrant transcript via nonsense-mediated mRNA decay (NMD). Collectively, these events account for ∼1800 human genetic diseases. Translational readthrough, the process by which near-cognate tRNAs decode stop codons and allow ribosomes to continue elongation beyond the stop codon, represents a possibility to suppress PTCs and restore full-length protein synthesis. Initially discovered in viruses as a mechanism to expand coding capacity, readthrough is now recognized as a regulated feature of eukaryotic gene expression influenced by both cis-acting sequence elements and trans-acting factors. Recent evidence highlights the remarkable context dependence of readthrough, revealing variation across transcripts, tissues, and developmental stages. In this review, we examine the molecular determinants that define stop codon recognition and readthrough efficiency, with particular emphasis on nucleotide context. We further discuss the mechanisms and binding sites of small molecules that promote PTC readthrough, and summarize the clinical development landscape of readthrough-inducing compounds for the treatment of diseases caused by nonsense mutations.

Humans↗
PubMed1994

Efficient targeting of foreign genes into the tobacco plastid genome.

The pPRV plasmids are vectors for targeted insertion of foreign genes into the tobacco plastid genome (ptDNA). The vectors are based on the pUC119 plasmid which replicates in E. coli but not in plastids. The spectinomycin resistance (aadA) gene and a multiple cloning site (MCS) are flanked by 1.8-kb and 1.2-kb ptDNA sequences. Biolistic delivery of vector DNA, followed by spectinomycin selection, yields plastid transformants at a reproducible frequency, approximately 1 transplastomic line per bombarded sample. The selected aadA gene and linked non-selectable genes cloned into the MCS are incorporated into the ptDNA by two homologous recombination events via the flanking ptDNA sequences. The transplastomes thus generated are stable, and are maternally transmitted to the seed progeny. The pPRV vector series targets insertions between the divergently transcribed trnV gene and the rps12/7 operon. The lack of readthrough transcription of appropriately oriented transgenes makes the vectors an ideal choice for the study of transgene promoter activity.

Base Sequence↗
PubMed1993

UGA codon context which spans three codons. Reversal by ms2i6A37 in tRNA, mutation in rpsD(S4) or streptomycin.

Mutant UGA codon contexts which previously have been identified at different positions in the lacI part of a fused lacIlacZ gene were characterized with respect to translational readthrough in another genetic surrounding at a constant location. Although readthrough levels are systematically higher in this new location the "tight/leaky" characteristics of these codon contexts are essentially fully determined by the two codons flanking the nonsense codon itself. Analysis of some UGA hybrid contexts shows that the contribution to the codon context character by the codon either at the 5'-side (CCA or AGC) or at the 3'-side (NGU) is independent of the nature of the codon at the other side of UGA if this codon is decoded by trpT(Su9) suppressor tRNA. In a trpT(Su9), miaA double mutant strain, which lacks the ms2i6A37 modification in this tRNA, suppression is decreased at all UGA contexts investigated. However, in one case the contribution to the codon context character by the determinant flanking at one side is negatively affected by the nature of the codon at the other side of UGA. Thus, the character of a nonsense codon context in this case results from both flanking codons acting in a co-operative manner with the tRNA reading the middle UGA codon. This negative context effect is counteracted by a rpsD12 (ribosomal protein S4) mutation or by a sublethal concentration of streptomycin in the growth medium. It is suggested that the ms2i6A37 base in trpT(Su9) suppressor tRNA increases the efficiency of this tRNA by protecting it from ribosomal proofreading which is induced by codon context.

Bacterial Proteins↗
PubMed2006

Nrd1 interacts with the nuclear exosome for 3' processing of RNA polymerase II transcripts.

The exosome complex is involved in multiple RNA processing and degradation pathways. How exosome is recruited to particular RNA substrates and then chooses between RNA processing and degradation modes remains unclear. We find that the RNA binding protein Nrd1, complexed with its partners Nab3, Sen1, and cap binding complex, physically interacts with the nuclear form of exosome. Nrd1 stimulates the RNA degradation activity of the exosome in vitro. However, Nrd1 can also block 3' to 5' degradation by the exosome at some Nrd1 binding sites. Nrd1 mutations share some phenotypes with exosome mutants, including increased readthrough transcription from several mRNA and sn/snoRNA genes. Therefore, Nrd1 may recruit exosome to RNA and influence the choice between processing and degradation. Since Nrd1 is known to bind RNA polymerase II and be important for sn/snoRNA 3' end processing, Nrd1 may link transcription and RNA 3' end formation with surveillance by the exosome.

Base Sequence↗
PubMed1985

The envelope-associated 22K protein of human respiratory syncytial virus: nucleotide sequence of the mRNA and a related polytranscript.

We recently determined that respiratory syncytial virus (strain A2) encodes a fourth unique envelope-associated virion protein that has molecular weight of approximately 24,000, as estimated by gel electrophoresis. The nucleotide sequence of the mRNA encoding this novel protein has now been determined from five cDNA clones, including three that contain the complete mRNA sequence. The complete mRNA sequence is 957 nucleotides, exclusive of polyadenylate, and contains two partially overlapping open reading frames. The 5'-proximal open reading frame is favored for utilization by the criteria of the location and sequence of its translational start site. Furthermore, the calculated molecular weight of the encoded protein, 22,153, is in agreement with the previous estimate of 24,000 for the authentic protein identified by hybrid selection and in vitro translation. The sequence of the predicted protein, now designated the 22K protein, contains 194 amino acids, is relatively hydrophilic, and appears to be the most basic of the respiratory syncytial virus proteins. The mRNA also contains a second, internal open reading frame which would encode a protein of 90 amino acids. However, no evidence for this translation product is known. The first nine nucleotides in the mRNA sequence, 5'-GGGGCAAAU, are identical to the conserved sequence identified previously at the 5' termini of seven other respiratory syncytial viral mRNAs; the sequence at the 3' end of the 22K mRNA, 5'. . . AGUUAUUU-polyadenylate, contains the elements of the previously identified 3'-terminal consensus sequence for respiratory syncytial virus mRNAs, AGUUAA(N)1-4-polyadenylate (P. L. Collins, Y. T. Huang, and G. W. Wertz, Proc. Natl. Acad. Sci. U.S.A. 81:7683-7687). In addition, we present and describe the intergenic sequence of a dicistronic RNA derived from readthrough of the F and 22K protein genes.

Amino Acid Sequence↗
PubMed1987

Differential effect of ATP concentration on synthesis of vesicular stomatitis virus leader RNAs and mRNAs.

Cleavage of the beta-gamma bond of ATP is required for wild-type (wt) vesicular stomatitis virus transcription in vitro. Recent findings have established that a domain-specific phosphorylation of the virus NS protein is necessary for activity. We report here that RNA synthesis catalyzed by purified standard wt virions responded cooperatively to various ATP concentrations, with half-maximal activity at approximately 500 microM. In contrast, mutant polR1 standard virions and wt defective interfering particles both showed conventional Michaelis-Menten kinetic profiles with Km values of approximately 143 and approximately 133 microM, respectively. The former synthesize readthrough products of the leader-N gene junction in addition to plus-strand leader RNA and mRNAs, whereas the latter synthesize only minus-strand leader RNA. The cooperative response of wt virus products, however, was specific to mRNAs; the small fraction of the total products corresponding to plus-strand leader approximated Michaelis-Menten behavior. Since the unique phenotype of the polR mutants correlates with the synthesis of replicationlike products in vitro, the affected ATP-requiring function most likely regulates both transcription and replication. We suggest that this mutated function involves phosphorylation of viral proteins.

Adenosine Triphosphate↗
PubMed2004

Evidence against a direct role for the Upf proteins in frameshifting or nonsense codon readthrough.

The Upf proteins are essential for nonsense-mediated mRNA decay (NMD). They have also been implicated in the modulation of translational fidelity at viral frameshift signals and premature termination codons. How these factors function in both mRNA turnover and translational control remains unclear. In this study, mono- and bicistronic reporter systems were used in the yeast Saccharomyces cerevisae to differentiate between effects at the levels of mRNA turnover and those at the level of translation. We confirm that upfDelta mutants do not affect programmed frameshifting, and show that this is also true for mutant forms of eIF1/Sui1p. Further, bicistronic reporters did not detect defects in translational readthrough due to deletion of the UPF genes, suggesting that their function in termination is not as general a phenomenon as was previously believed. The demonstration that upf sui1 double mutants are synthetically lethal demonstrates an important functional interaction between the NMD and translation initiation pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Bacterial SsrA system plays a role in coping with unwanted translational readthrough caused by suppressor tRNAs.

BACKGROUND: Bacterial SsrA RNA (also known as tmRNA or 10Sa RNA) mediates the addition of a short peptide tag to the C-terminus of the nascent polypeptide when a ribosome is stalled at the 3' end of an mRNA lacking a stop codon. This process, called trans-translation, rescues the stalled ribosome and ensures degradation of tagged polypeptides by ATP-dependent proteases. To fully understand the physiological roles of SsrA RNA, it is essential to know how endogenous mRNA targets for the SsrA system are generated in cells. The aim of the present study is to examine how translational readthrough by suppressor tRNAs affects trans-translation in Escherichia coli. RESULTS: We demonstrated that SsrA tagging of bulk cellular proteins was significantly enhanced by an ochre or an amber suppressor tRNA. Western blot analysis of proteins produced from specific genes possessing a Rho-independent terminator revealed that readthrough at the normal stop codon leads to an efficient tagging and proteolysis of the extended proteins. Size analyses of both protein and mRNA suggested that tagging of extended proteins occurs because ribosome passing through the normal stop codon presumably reach the 3' end of mRNA defined by the transcription terminator hairpin. The inhibitory effect of ssrA mutation on cell growth was markedly amplified in cells with an ochre suppressor tRNA. CONCLUSION: The present finding suggests that the SsrA system contributes to scavenge errors and/or problems caused by translational readthrough that occurs typically in the presence of a suppressor tRNA.

Amino Acid Sequence↗
PubMed1997

Cloning and overexpression of polypeptide release factor 1 of Thermus thermophilus.

A prfA gene encoding polypeptide release factor RF1 was cloned from Thermus thermophilus. T thermophilus RF1 shares 68% homology with Escherichia coli RF1, and its overproduction reduced readthrough translation of UAG, not of UGA, in the lacZ gene. Rapid purification of T thermophilus RF1 was achieved by T7-RNA polymerase driven overexpression of T thermophilus RF1 protein with a C-terminal histidine tag.

Amino Acid Sequence↗
PubMed1986

Characterization of clones for the sixth (L) gene and a transcriptional map for morbilliviruses.

cDNA clones of the largest RNA transcript of the canine distemper and measles morbilliviruses were characterized. This presumably codes for the L protein of these viruses. mRNA 4 was identified as coding for the haemagglutinin protein of measles virus. From an analysis of readthrough transcripts representing tandem copies of two or three genes we established a transcriptional map and the gene order on the negative strand genome of the morbilliviruses to be 3'-N-P + C-M-F-H-L-5'. The data exclude the presence of small intervening genes between the six major genes of morbilliviruses and indicate the gene order to be similar to that of Sendai virus and different from that of simian virus 5.

Cloning, Molecular↗
PubMed2004

Translation termination in Arabidopsis thaliana: characterisation of three versions of release factor 1.

Translation termination is mediated in all eukaryotes by the two release factors eRF1 and eRF3. Most organisms have a single eRF1 gene, however, three isogenes of eRF1 are found in Arabidopsis thaliana. They have no introns in the coding region which may indicate that some are pseudogenes. However, each was expressed and able to rescue a temperature sensitive eRF1-mutant of Saccharomyces cerevisiae indicating functional redundancy in A. thaliana. While normally a highly accurate process, translation termination can be directed to fail by sequence elements within an messenger RNA (mRNA). Interestingly, a well-characterised readthrough element follows the stop codon in one of these three isogenes (designated eRF1-1). This element was shown to be capable of inducing readthrough in an in vitro assay using a dual luciferase reporter, but surprisingly readthrough could not be detected using the complete gene context. The results highlight the diversity and duplication of genes within plant genomes, but also emphasize the conservation of the translation process across kingdoms.

Amino Acid Motifs↗
PubMed1991

Expression of lacZ gene fusions affects downstream transcription in yeast.

Chimeric genes containing Escherichia coli lacZ sequences are often used to characterize gene expression in yeast cells. By Northern analysis, we found that such genes produce multiple transcripts due to inefficient 3'-end formation. The same transcript pattern was found for two related chimeric genes when these genes were cloned separately into the commonly used vector, YIp5, and integrated into the yeast genome at two different locations. Each chimeric gene was composed of promoter and N-terminal coding regions from the yeast SSA1 or SSA2 genes fused in-frame to the lac operon. Transcripts were shown to initiate within the yeast promoter fragment, but transcript size indicated that 3' ends were localized to three different regions: within the lac operon near the 3' end of the lacZ gene; near a terminator region previously identified upstream of the URA3 gene in YIp5; and at the URA3 terminator region. Readthrough transcription of the URA3 promoter from upstream lac sequences decreased the basal activity of the URA3 promoter, although induced URA3 transcription levels were unaffected. This readthrough transcription also resulted in a novel, longer URA3 transcript.

Base Sequence↗
PubMed2006

The gene of an archaeal alpha-L-fucosidase is expressed by translational frameshifting.

The standard rules of genetic translational decoding are altered in specific genes by different events that are globally termed recoding. In Archaea recoding has been unequivocally determined so far only for termination codon readthrough events. We study here the mechanism of expression of a gene encoding for a alpha-l-fucosidase from the archaeon Sulfolobus solfataricus (fucA1), which is split in two open reading frames separated by a -1 frameshifting. The expression in Escherichia coli of the wild-type split gene led to the production by frameshifting of full-length polypeptides with an efficiency of 5%. Mutations in the regulatory site where the shift takes place demonstrate that the expression in vivo occurs in a programmed way. Further, we identify a full-length product of fucA1 in S.solfataricus extracts, which translate this gene in vitro by following programmed -1 frameshifting. This is the first experimental demonstration that this kind of recoding is present in Archaea.

Archaea↗
PubMed2000

Analysis of iceA1 transcription in Helicobacter pylori.

BACKGROUND: Transcription of the Helicobacter pylori iceA1 gene is induced following adherence of the bacterium to gastric epithelial cells in vitro, suggesting that this gene might be involved in H. pylori pathogenesis. Consequently, the current studies were undertaken to characterize iceA1 transcription and to define the structure of iceA1-containing transcripts to evaluate the potential of this gene to encode functional proteins. MATERIALS AND METHODS: Northern blots and primer extension of RNA isolated from broth-grown cultures of various H. pylori strains was done to analyze iceA1-specific gene transcription. Reverse transcriptase (RT)-PCR was used to determine the levels of iceA1 transcripts derived from readthrough transcription that was initiated upstream of iceA1 within the 5'-flanking cysE gene. RESULTS: Three major transcripts were detected and each was initiated from a common promoter, designated PI. Two of these transcripts were comprised of iceA1 sequence, while a third transcript was dicistronic and included the downstream gene, hpyIM. In addition, 10-fold lower levels of iceA1 transcripts were initiated upstream of PI, either within or immediately downstream of cysE. CONCLUSIONS: The present analysis suggests that iceA1 does not encode a functional protein in the majority of H. pylori strains. However, transcription of hpyIM, which encodes a highly conserved DNA adenine methyltransferase, is linked to iceA1 transcription. Therefore, iceA1 may affect H. pylori virulence in vivo through transcriptional regulation of hpyIM expression levels, which may result in specific variations in DNA methylation patterns leading to alteration in the expression of genes involved in virulence or pathogenesis.

Acetyltransferases↗
PubMed2006

Readthrough acetylcholinesterase expression remains minor after stress or exposure to inhibitors.

The gene of mammalian acetylcholinesterase (AChE) generates multiple molecular forms, by alternative splicing of its transcripts and association of the tailed variant (AChET) with structural proteins. In the mammalian brain, the major AChE species consists of AChET tetramers anchored to the cell membrane of neurons by the PRiMA protein (Perrier et al., 2002). Stress and anticholinesterase inhibitors have been reported to induce rapid and long-lasting expression of the readthrough variant (AChER) in the mouse brain (Kaufer et al., 1998). In the readthrough transcript, there is no splicing after the last exon encoding the catalytic domain, so that the entire alternatively spliced 3' region is maintained. It encodes a C-terminal peptide with no specific interaction properties: COS cells transfected with AChER produce a soluble, nonamphiphilic monomeric form. We quantified AChER and total AChE expression in the mouse brain after an immobilization stress and after heat shock in neuroblastoma cells, and compared the observed effects with those induced by irreversible AChE inhibition (Perrier et al., 2005).

Acetylcholinesterase↗
PubMed2004

Genetic reprogramming by retroviruses: enhanced suppression of translational termination.

Viruses often exploit or subvert host machinery for their own purposes during replication. A search for proteins interacting with the murine leukemia virus reverse transcriptase (RT) recently provided a new example of such exploitation. RT was found to bind the eukaryotic translational release factor 1 (eRF1), the protein that recognizes stop codons and, in complex with eRF3, causes termination and polypeptide release from the ribosome. RT is derived from a large Gag-Pol polyprotein, and its synthesis requires a translational readthrough, a suppression of termination, at a stop codon at the end of the gag gene. The binding of eRF1 by RT was found to inhibit eRF1 action, enhance the efficiency of readthrough, and thus cause higher levels of RT synthesis. The observations suggest that retroviruses manipulate the translational machinery in sophisticated ways to fine-tune their own gene expression.

Codon, Terminator↗
PubMed1986

Expression of the Plasmodium knowlesi circumsporozoite antigen in Escherichia coli directed by Plasmodium bacterial-like promoter sequences.

The Plasmodium knowlesi circumsporozoite (CS) gene is expressed in Escherichia coli directly from a parasite genomic DNA fragment, using promoter and ribosome-binding site (RBS) sequences present in this fragment. Transcription of the CS gene in E. coli is directed by tandem Plasmodium bacterial-like promoter elements located within the 0.5-kb EcoRI-HindIII fragment roughly 2.5 kb 5' from the CS gene within the 11-kb EcoRI parasite genomic DNA fragment. No readthrough from vector promoters or fortuitous promotion from plasmodial A + T-rich sequences was observed. The endogenous Plasmodium promoter of the CS gene does not seem to be recognized by E. coli RNA polymerases. Two tandem E. coli-recognized promoters are relatively strong judging by their ability to drive the bacterial chloramphenicol acetyl-transferase (CAT) gene. Translation of the message must be achieved by utilising an AAGAA sequence 4 bp 5' from the ATG initiation codon as RBS.

Animals↗