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PubMed1990

A single mutation in 16S rRNA that affects mRNA binding and translation-termination.

A single base change in 16S rRNA (C726 to G) has previously been shown to have a dramatic effect on protein synthesis in E. coli (1). This paper more specifically details the effects of the mutation on mRNA binding and translation-termination. The in vitro technique of toeprinting (2) was used to demonstrate that 30S subunits containing the mutation 726G had an altered binding affinity for mRNA by comparison to the wild type. In addition, expression of the mutant ribosomes in vivo resulted in exclusive suppression of the UGA nonsense codon. This effect was supported by in vitro studies that showed the mutant ribosomes to have an altered binding affinity for Release Factor-2.

Base Sequence↗
PubMed1994

Positions 13 and 914 in Escherichia coli 16S ribosomal RNA are involved in the control of translational accuracy.

Using a conditional expression system with the temperature-inducible lambda PL promoter, we previously showed that the single mutations 13U-->A and 914A-->U, and the double mutation 13U-->A and 914A-->U in Escherichia coli 16S ribosomal RNA impair the binding of streptomycin (Pinard et al., The FASEB Journal, 1993, 7, 173-176). In this study, we found that the two single mutations and the double mutation increase translational fidelity, reducing in vivo readthrough of nonsense codons and frameshifting, and decreasing in vitro misincorporation in a poly(U)-directed system. Using oligodeoxyribonucleotide probes which hybridize to the 530 loop and to the 1400 region of 16S rRNA, two regions involved in the control of tRNA binding to the A site, we observed that the mutations in rRNA increase the binding of the probe to the 530 loop but not to the 1400 region. We suggest that the mutations at positions 13 and 914 of 16S rRNA induce a conformational rearrangement in the 530 loop, which contributes to the increased accuracy of the ribosome.

Base Sequence↗
PubMed1997

In vitro suppression as a tool for the investigation of translation initiation.

An in vitro protein synthesizing system that employs rabbit reticulocyte lysates has been employed for protein production from mRNAs containing nonsense (UAG) codons in the presence of misacylated suppressor tRNAs.The system includes a misacylated Escherichia coli tRNAAlaCUA that functions at least as efficiently as any suppressor tRNA transcript reported to date and which has been shown not to be a substrate for (re)activation by alanyl-tRNA synthetase. Application of the optimized system for preparation of dihydrofolate analogs has also permitted analysis of competing mechanisms that control the sites(s) of translation initiation.

Amino Acid Sequence↗
PubMed1998

Identity elements and aminoacylation of plant tRNATrp.

Mutation of the Arabidopsis thaliana tRNA (Trp)(CCA) anticodon or of the A73 discriminator base greatly diminishes in vitro aminoacylation with tryptophan, indicating the importance of these nucleotides for recognition by the plant tryptophanyl-tRNA synthetase. Mutation of the tRNA (Trp)(CCA) anticodon to CUA so as to translate amber nonsense codons permits tRNA (Trp)(CCA) to be aminoacylated by A.thaliana lysyl-tRNA synthetase. Thus, translational suppression by tRNA (TRP)(CCA) observed in plant cells includes significant incorporation of lysine into protein.

Animals↗
PubMed1980

Structure of a promotor on plasmid pMB9 derived from plasmid pSC101.

The DNA sequence of a 354 basepair EcoRI-HindIII fragment of plasmid pMB9 which has originally been derived from plasmid pSC101 has been resolved. This fragment contains a promoter for transcription directed towards the EcoRI site. Escherichia coli RNA polymerase binds to a region within the EcoRI-HindIII fragment which contains the heptamer 5' TATGGTG (132-126) and the duodecamer 5' TGATGAACATCA (158-147). Based on commonalities with other promotors these DNA sequences probably function as, respectively, "binding site" and "recognition site". Furthermore, this fragment harbours a translation reading frame free of nonsense codons and at about 25 basepairs from the indicated heptamer a nucleotide sequence which meets with the requirements for initiation of translation. By heteroduplex mapping it was shown that the EcoRI-HindIII fragment has been derived from a region near or within the origin of replication of pSC101. The copynumber of plasmids containing the EcoRI-HindIII fragment is two-fold lower than that of plasmids lacking this fragment. This effect might be related to the original function of this fragment on plasmid pSC101.

Base Sequence↗
PubMed2003

The complete nucleotide sequence of the hornwort (Anthoceros formosae) chloroplast genome: insight into the earliest land plants.

It is generally believed that bryophytes are the earliest land plants. However, the phylogenetic relationships among bryophytes, including mosses, liverworts and hornworts, are not clearly resolved. To obtain more information on the earliest land plants, we determined the complete nucleotide sequence of the chloroplast genome from the hornwort Anthoceros formosae. The circular double-stranded DNA of 161 162 bp is the largest genome ever reported among land plant chloroplasts. It contains 76 protein, 32 tRNA and 4 rRNA genes and 10 open reading frames (ORFs), which are identical with the chloroplast genome of the other green plants analyzed. The major difference is a larger inverted repeat than that of the liverwort Marchantia, Anthoceros contains an excess of ndhB and rps7 genes and the 3' exon of rps12. The genes matK and rps15, commonly found in the chloroplast genomes of land plants, are pseudogenes. The intron of rrn23 is the first finding in the known chloroplast genomes of land plants. A striking feature of the hornwort chloroplast is that more than half of the protein-coding genes have nonsense codons, which are converted into sense codons by RNA editing. Maximum-likelihood (ML) analysis, based on 11 518 amino acid sites of 52 proteins encoded in the chloroplast genomes of the green plants, placed liverworts as the sister to all other land plants.

DNA, Chloroplast↗
PubMed2003

RNA editing in hornwort chloroplasts makes more than half the genes functional.

RNA editing in chloroplasts alters the RNA sequence by converting C-to-U or U-to-C at a specific site. During the study of the complete nucleotide sequence of the chloroplast genome from the hornwort Anthoceros formosae, RNA editing events have been systematically investigated. A total of 509 C-to-U and 433 U-to-C conversions are identified in the transcripts of 68 genes and eight ORFs. No RNA editing is seen in any of the rRNA but one tRNA suffered a C-to-U conversion at an anticodon. All nonsense codons in 52 protein-coding genes and seven ORFs are removed in the transcripts by U-to-C conversions, and five initiation and three termination codons are created by C-to-U conversions. RNA editing in intron sequence suggests that editing can precede intercistronic processing. The sequence complementary to the edited site is proposed as a distant cis-recognition element.

Amino Acid Substitution↗
PubMed2003

The Upf-dependent decay of wild-type PPR1 mRNA depends on its 5'-UTR and first 92 ORF nucleotides.

mRNAs containing premature translation termination codons (nonsense mRNAs) are targeted for deadenylation-independent degradation in a mechanism that depends on Upf1p, Upf2p and Upf3p. This decay pathway is often called nonsense- mediated mRNA decay (NMD). Nonsense mRNAs are decapped by Dcp1p and then degraded 5' to 3' by Xrn1p. In the yeast Saccharomyces cerevisiae, a significant number of wild-type mRNAs accumulate in upf mutants. Wild-type PPR1 mRNA is one of these mRNAs. Here we show that PPR1 mRNA degradation depends on the Upf proteins, Dcp1p, Xrn1p and Hrp1p. We have mapped an Upf1p-dependent destabilizing element to a region located within the 5'-UTR and the first 92 bases of the PPR1 ORF. This element targets PPR1 mRNA for Upf-dependent decay by a novel mechanism.

5' Untranslated Regions↗
PubMed2003

Initiation-mediated mRNA decay in yeast affects heat-shock mRNAs, and works through decapping and 5'-to-3' hydrolysis.

The degradation of mRNA in the yeast Saccharomyces cerevisiae takes place through several related pathways. In the most general mRNA-decay pathway, that of poly(A)-dependent decay, the normal shortening of the poly(A) tail on an mRNA molecule by deadenylation triggers mRNA decapping by the enzyme Dcp1p, followed by exonucleolytic digestion by Xrn1p. A specialized mRNA-decay pathway, termed nonsense-mediated decay, comes into play for mRNAs that contain an early nonsense codon. This pathway operates through the Upf proteins in addition to Dcp1p and Xrn1p. Previously, we identified a different specialized mRNA-decay pathway, the initiation-mediated decay pathway, and showed that it affects two Hsp70 heat-shock mRNAs under conditions of slowed translation initiation. Here we report that initiation-mediated mRNA decay also works through the Dcp1 and Xrn1 enzymes, and requires ongoing transcription by RNA polymerase II. We show that several other heat-shock mRNAs, including two from the Hsp90 gene family and three more from the Hsp70 gene family, are also subject to initiation-mediated decay, whereas a variety of non-heat-shock mRNAs are not affected.

Adaptor Proteins, Signal Transducing↗
PubMed1988

Fitness effects of amino acid replacements in the beta-galactosidase of Escherichia coli.

Two genetic procedures were used to obtain amino acid replacements in the lacZ-encoded beta-galactosidase in Escherichia coli. Amino acid replacements could be obtained without regard to their effects on lactase activity by selecting spontaneous mutations that relieved the strong polarity of six nonsense mutations. When streaked on MacConkey-lactose indicator plates, approximately 75% of these mutants gave strong red lactose-fermenting colonies, and 25% gave white nonfermenting colonies. Mutants from 11 other nonsense codons were isolated directly using MacConkey-lactose indicator plates, on which positive color indication requires only 0.5% of the wildtype lactase activity. Among the total of 17 codons, 25 variant beta-galactosidases were identified using electrophoresis and thermal denaturation studies. The fitness effects of these variant beta-galactosidases were determined using competition experiments conducted with lactose as the sole nutrient limiting the growth rate in chemostat cultures. Three of the replacements were deleterious, one was selectively advantageous, and the selective effects of the remaining 21 were undetectable under conditions in which the smallest detectable selection coefficient was approximately 0.4%/generation.

Alleles↗
PubMed1988

Selenocysteine's mechanism of incorporation and evolution revealed in cDNAs of three glutathione peroxidases.

The nonsense codon, UGA, has for the first time recently been shown to encode selenocysteine in two proteins, mouse glutathione peroxidase (GSH-Px) (EC 1.11.1.9) and bacterial formate dehydrogenase. A co-translational rather than post-translational selenium-incorporation mechanism has been implicated. Furthermore, high expression levels of GSH-Px have suggested that suppression of termination is efficient and specific. We have isolated and characterized pituitary, kidney and placenta cDNAs for bovine, human and mouse GSH-Px respectively. It is demonstrated that this novel suppression event occurs in diverse tissues, in at least three mammalian species and at the translational step. Surprisingly, GSH-Px is shown to be extramitochondrially encoded, indicating a cytosolic suppression event rather than one utilizing the mitochondria's well-documented extended codon-reading ability. Sequence analysis reveals that a simple proximal contextual pattern responsible for readthrough does not exist. Analysis of predicted secondary structures of mRNAs, however, has revealed a conformation which may be unique to selenocysteine proteins and may prove useful as a tool for artificial incorporation of selenocysteines. A human intron for GSH-Px from an unspliced mRNA has been isolated whose position indicates an ancient, divergent evolutionary relationship with thioredoxin-S2, rather than an independent convergent one.

Animals↗
PubMed1993

Altering substrate preference of carboxypeptidase Y by a novel strategy of mutagenesis eliminating wild type background.

To change the substrate preference of carboxypeptidase Y the putative substrate binding pocket was subjected to random mutagenesis. Based upon the three-dimensional structure of a homologous enzyme from wheat, we hypothesized that Tyr147, Leu178, Glu215, Arg216, Ile340 and Cys341 are the amino acid residues of carboxypeptidase Y that constitute S1, the binding pocket for the penultimate amino acid side chain of the substrate. We developed a new and generally applicable mutagenesis strategy to facilitate efficient screening of a large number of mutants with multiple changes in carboxypeptidase Y. The key feature is the elimination of wild type background by introducing a nonsense codon at each target site for subsequent mutagenesis by degenerate oligonucleotides. The entire hypothesized S1 binding pocket and subsets of it were subjected to saturation mutagenesis by this strategy, and screening yielded a number of mutant enzymes which have up to 150 times more activity (kcat/Km) towards CBZ-Lys-Leu-OH than the wild type enzyme. All selected mutants with increased activity have mutations at position 178. Mutagenesis of positions 215 and 216 has virtually no effect on the activity, while mutating positions 340 and 341 generally reduces activity.

Amino Acids↗
PubMed1999

A GLC1A gene Gln368Stop mutation in a patient with normal-tension open-angle glaucoma.

PURPOSE: To present a case involving a patient with normal-tension glaucoma with a Gln368Stop mutation of the myocilin/trabecular meshwork inducible glucocorticoid response protein (MYOC/TIGR) gene. METHODS: Slit-lamp biomicroscopic and gonioscopic examination, morphometry of the optic disc, 24-hour intraocular pressure (IOP) profile, and perimetry were performed to determine the phenotype of the patient. Neurologic examination and a computed tomographic (CT) scan of the brain were performed to rule out a neurologic disorder. Single-strand confirmation polymorphism (SSCP) analysis and subsequent sequence analysis of blood was performed for genotyping of the GLC1A gene. RESULTS: A nonsense codon, namely a Gln368Stop mutation in the third exon of the GLC1A gene, was found in this patient with normal-tension glaucoma. CONCLUSION: In contrast to previous reports, a Gln368Stop mutation of the GLC1A gene need not be confined to patients with glaucomatous optic atrophy due to high IOP. The pathogenesis of glaucoma associated with GLC1A gene mutations might be more complex than expected, and (unknown) suppressor mechanisms have to be considered.

Adult↗
PubMed2000

Invasiveness in chickens, stress resistance and RpoS status of wild-type Salmonella enterica subsp. enterica serovar typhimurium definitive type 104 and serovar enteritidis phage type 4 strains.

The heat and acid resistance and the ability to survive airdrying on commonly used kitchen surfaces were assessed for clinical and environmental strains of Salmonella enterica subsp. enterica serovar Typhimurium, definitive type (DT) 104. Three out of thirty-eight strains of DT 104 were found to be more sensitive in stationary phase to the stresses examined than the other strains. This compares to a previous study by the authors which showed that seven out of forty serovar Enteritidis phage type (PT) 4 strains were more sensitive. RpoS activity was examined indirectly in selected strains of DT 104 and PT 4. In those with normal stress resistance a 100-fold induction of an RpoS-dependent spvR/A:'::luxCDABE fusion was observed upon entry into stationary phase. The sensitive strains examined showed either no induction or a reduced level of spvR/A:'::luxCDABE expression. The rpoS gene was sequenced from these strains and three were found to harbour mutations including one deletion, one base-pair substitution resulting in a nonsense codon, and one insertion causing a frameshift resulting in an early stop codon. Strains with negligible or reduced spvR/A:'::luxCDABE expression had low stress resistance. All strains of DT 104 could be recovered from liver and spleen tissues of infected hens 14 d post-infection, but one with no induction of spvR/A:'::luxCDABE expression was significantly less likely to be recovered from chicken reproductive tissues, liver or spleen than the majority of other strains, including one with reduced spvR/A:'::luxCDABE expression. This work has demonstrated that clinical and environmental strains of DT 104 and PT 4 not infrequently harbour mutations in the rpoS allele. It is possible that the rpoS mutations may have occurred during the initial isolation of the strains. The ability of a strain to cause infection, however, also depends on factors such as host susceptibility and dose.

Animals↗
PubMed1998

The surveillance complex interacts with the translation release factors to enhance termination and degrade aberrant mRNAs.

The nonsense-mediated mRNA decay pathway is an example of an evolutionarily conserved surveillance pathway that rids the cell of transcripts that contain nonsense mutations. The product of the UPF1 gene is a necessary component of the putative surveillance complex that recognizes and degrades aberrant mRNAs. Recent results indicate that the Upf1p also enhances translation termination at a nonsense codon. The results presented here demonstrate that the yeast and human forms of the Upf1p interact with both eukaryotic translation termination factors eRF1 and eRF3. Consistent with Upf1p interacting with the eRFs, the Upf1p is found in the prion-like aggregates that contain eRF1 and eRF3 observed in yeast [PSI+] strains. These results suggest that interaction of the Upf1p with the peptidyl release factors may be a key event in the assembly of the putative surveillance complex that enhances translation termination, monitors whether termination has occurred prematurely, and promotes degradation of aberrant transcripts.

Fungal Proteins↗
PubMed1997

Intracellular carbonic anhydrase is essential to photosynthesis in Chlamydomonas reinhardtii at atmospheric levels of CO2. Demonstration via genomic complementation of the high-CO2-requiring mutant ca-1.

Genomic complementation of the high-CO2-requiring mutant ca-1-12-1C of Chlamydomonas reinhardtii was achieved by transformation with DNA pools from an indexed cosmid library of wild-type genomic DNA. Transformation of mutant cells with cosmid DNA from two microtiter plates in the library produced colonies that grew phototrophically at atmospheric CO2 levels. Transformations with cosmid DNA from each of the rows and files of the two plates pinpointed one well in each plate with a cosmid bearing the targeted gene. Sequencing of cosmid subclones revealed a gene encoding a recently identified C. reinhardtii chloroplast carbonic anhydrase (CAH3). Transformations with chimeric constructs combining different portions of the wild-type and mutant genes indicated the presence of a mutation in the 5'-half of the gene. Comparison of mutant and wild-type gene sequences in this region revealed a G-to-A substitution in the mutant gene, which produced a nonsense codon. The data presented demonstrate that the carbonic anhydrase produced from the CAH3 gene is essential to the inorganic carbon-concentrating mechanism in C. reinhardtii and that genomic complementation can be a facile and efficient means for isolating genes associated with defects affecting photosynthesis and other physiological processes in this eukaryotic green alga.

Amino Acid Sequence↗
PubMed1997

A deletion in the PHYD gene of the Arabidopsis Wassilewskija ecotype defines a role for phytochrome D in red/far-red light sensing.

The PHYD gene of the Wassilewskija (Ws) ecotype of Arabidopsis contains a 14-bp deletion (the phyD-1 mutation) beginning at amino acid 29 of the reading frame, resulting in translation termination at a nonsense codon 138 nucleotides downstream of the deletion end point. Immunoblot analyses showed that Ws lacks phyD but contains normal levels of phyA, phyB, and phyC. By backcrossing into the Ws and Landsberg erecta genetic backgrounds, we constructed sibling pairs of PHYD+ and phyD-1 lines and of phyB- PHYD+ and phyB- phyD- lines. Hypocotyl lengths after growth under white or red light increased sequentially in strains that were B+D+, B+D-, B-D+, and B-D-. In the Ws genetic background, an increase in petiole length, a reduction in cotyledon area and in anthocyanin accumulation in seedling stems, a diminished effect of an end-of-day pulse of far-red light on hypocotyl elongation, and a decrease in the number of rosette leaves at the onset of flowering were also seen sequentially in these lines. Thus, phyD, which is approximately 80% identical in amino acid sequence to phyB, acts in conjunction with phyB in regulating many shade avoidance responses. The existence of the apparently naturally occurring phyD-1 mutation indicates that phyD is not essential in some natural environments.

Apoproteins↗