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PubMed1995

5' contexts of Escherichia coli and human termination codons are similar.

The nearest 5' context of 2559 human stop codons was analysed in comparison with the same context of stop-like codons (UGG, UGC, UGU, CGA for UGA; CAA, UAU, UAC for UAA; and UGG, UAU, UAC, CAG for UAG). The non-random distribution of some nucleotides upstream of the stop codons was observed. For instance, uridine is over-represented in position -3 upstream of UAG. Several codons were shown to be over-represented immediately upstream of the stop codons: UUU(Phe), AGC(Ser), and the Lys and Ala codon families before UGA; AAG(Lys), GCG(Ala), and the Ser and Leu codon families before UAA; and UCA(Ser), AUG(Met), and the Phe codon family before UAG. In contrast, the Thr and Gly codon families were under-represented before UGA, while ACC(Thr) and the Gly codon family were under-represented before UAG and UAA respectively. In an earlier study, uridine was shown to be over-represented in position -3 before UGA in Escherichia coli [Arkov,A.L., Korolev,S.V. and Kisselev,L.L. (1993) Nucleic Acids Res., 21,2891-2897]. In that study, the codons for Lys, Phe and Ser were shown to be over-represented immediately upstream of E. coli stop codons. Consequently, E. coli and human termination codons have similar 5' contexts. The present study suggests that the 5' context of stop codons may modulate the efficiency of peptide chain termination and (or) stop codon readthrough in higher eukaryotes, and that the mechanisms of such a modulation in prokaryotes and higher eukaryotes may be very similar.

Amino Acids↗
PubMed2004

Complete set of orthogonal 21st aminoacyl-tRNA synthetase-amber, ochre and opal suppressor tRNA pairs: concomitant suppression of three different termination codons in an mRNA in mammalian cells.

We describe the generation of a complete set of orthogonal 21st synthetase-amber, ochre and opal suppressor tRNA pairs including the first report of a 21st synthetase-ochre suppressor tRNA pair. We show that amber, ochre and opal suppressor tRNAs, derived from Escherichia coli glutamine tRNA, suppress UAG, UAA and UGA termination codons, respectively, in a reporter mRNA in mammalian cells. Activity of each suppressor tRNA is dependent upon the expression of E.coli glutaminyl-tRNA synthetase, indicating that none of the suppressor tRNAs are aminoacylated by any of the twenty aminoacyl-tRNA synthetases in the mammalian cytoplasm. Amber, ochre and opal suppressor tRNAs with a wide range of activities in suppression (increases of up to 36, 156 and 200-fold, respectively) have been generated by introducing further mutations into the suppressor tRNA genes. The most active suppressor tRNAs have been used in combination to concomitantly suppress two or three termination codons in an mRNA. We discuss the potential use of these 21st synthetase-suppressor tRNA pairs for the site-specific incorporation of two or, possibly, even three different unnatural amino acids into proteins and for the regulated suppression of amber, ochre and opal termination codons in mammalian cells.

Amino Acyl-tRNA Synthetases↗
PubMed2002

flaA-like sequences containing internal termination codons (TAG) in urease-positive thermophilic Campylobacter isolated in Japan.

AIMS: To demonstrate two flaA-like sequences containing two internal termination codons (TAG) in two Japanese strains of urease-positive thermophilic Campylobacter (UPTC). METHODS AND RESULTS: A primer pair of A1 and A2, which ought to generate a product of approx. 1700 bp of the flaA gene for Campylobacter jejuni, was used to amplify products of approx. 1450 bp for two Japanese strains of UPTC, CF89-12 and CF89-14. After molecular cloning and sequencing, the nucleotide sequences of the amplicons from the two strains were found to be 1461 bp in length and to have nucleotide sequence differences in relation to each other at four nucleotide positions, respectively. CONCLUSIONS: Nucleotide and amino acid sequence alignment and homology analysis demonstrated that the polymerase chain reaction (PCR) amplicons from the two Japanese strains have approx. 83% nucleotide and 80% amino acid sequence homology to the possible open reading frame of the flaA gene of UPTC NCTC 12892. SIGNIFICANCE AND IMPACT OF THE STUDY: Surprisingly, both PCR amplicons from the Japanese UPTC have two internal termination codons (TAG) at nucleotide positions from 775 to 777 and 817 to 819, respectively.

Amino Acid Sequence↗
PubMed1989

Differential use of termination codons in ciliated protozoa.

Sequence analysis of genes in four species of ciliated protozoa and analysis of tRNAs in Tetrahymena has demonstrated that TAG and TAA encode glutamine or glutamic acid in these organisms and TGA is the only stop codon. Thus, it has generally been assumed that all ciliates use a nonuniversal genetic code in which TGA acts as the sole termination codon. We have sequenced the linear DNA molecules that carry an actin gene and a beta-tubulin gene from the ciliate Euplotes crassus. These genes are shown to use TAA as a termination codon based on homology to known actin and beta-tubulin gene sequences. In addition, we have sequenced a portion of the 3' terminus of the E. crassus H4 histone gene and show that it also uses TAA as a termination codon. These data indicate that the timing of genetic code changes in the ciliates must be reconsidered.

Actins↗
PubMed1982

Nucleotide sequence and evolution of the rat mitochondrial cytochrome b gene containing the ochre termination codon.

The nucleotide sequences of the genes for cytochrome b and three potential transfer RNAs (tRNAPro, tRNAThr and tRNAGlu) in cloned rat mitochondrial DNA were determined. The derived amino acid sequence of the cytochrome b protein from the light strand indicated that the C-terminal amino acid is asparagine and the ochre termination codon is encoded in the DNA, in contrast to the the lack of termination codon in the reading frame of human [Anderson et al., Nature 290 (1981) 457] or mouse [Bibb et al., Cell 26 (1981) 167] mitochondrial DNA. The first ATG codon of the cytochrome b gene was spaced five nucleotides from the 5'-end of the tRNAGlu gene on the heavy strand. There was a single nucleotide spacing between the termination codon of the cytochrome b gene and the 5' end of the tRNAThr gene in the light strand. There was also a single nucleotide spacing between the 3'-end of the tRNAThr gene and the 3'-end of the tRNAPro gene on the heavy strand. The amino acid and nucleotide sequences of the cytochrome b genes of mammals and yeast [Nobrega and Tzagoloff, J. Biol. Chem. 255 (1980) 9828] were compared to reveal structural differences in two very different species. At the same time, amino acid substitutions in particular regions of the mammalian gene corresponding to the exon-intron boundaries in the yeast gene were noted. These genetic features are discussed in relation to the extreme compression of genetic information in the mammalian mitochondrial genome as related to the evolution of the gene organization and its sequence.

Amino Acid Sequence↗
PubMed1998

Novel compound heterozygous mutations in the plectin gene in epidermolysis bullosa with muscular dystrophy and the use of protein truncation test for detection of premature termination codon mutations.

Absence of plectin, a large cytoskeleton-associated protein expressed in the skin and muscle, has been shown to underlie epidermolysis bullosa with muscular dystrophy (EB-MD), an autosomal recessive disorder (OMIM No. 226670). In the present study, we report the case of a patient who presented with neonatal blistering and late-onset muscular dystrophy with nail and tooth abnormalities, as well as severe mucocutaneous involvement including laryngeal webs and urethral strictures, features not previously reported in this syndrome. Mutation detection, based on the use of heteroduplex analysis, revealed that the proband was a compound heterozygote for two plectin mutations, 4416delC/4359ins13, both resulting in premature termination codons in the plectin rod domain. Because these mutations, and the majority of those previously reported, reside within exon 32 of the plectin gene (PLEC1), we applied the protein truncation test (PTT) to screen for mutations in the two large 3' exons (nos. 32 and 33) of PLEC1, which together comprise approximately 75% of the coding region of the gene. PTT readily detected truncated polypeptides in the proband profiled in this study, as well as in a patient in whom we have previously identified premature termination codon mutations in exon 32. Thus, PTT provides a rapid and reliable strategy to identify premature termination codon mutations from genomic DNA within PLEC1.

Adult↗
PubMed1981

Usage of the three termination codons in a single eukaryotic cell, the Xenopus laevis oocyte.

Oocytes from Xenopus laevis were injected with purified amber (UAG), ochre (UAA), and opal (UGA) suppressor tRNAs from yeasts. The radioactively labeled proteins translated from the endogenous mRNAs were then separated on two-dimensional gels. All three termination codons are used in a single cell, the Xenopus laevis oocyte. But a surprisingly low number of readthrough polypeptides were observed from the 600 mRNAs studied in comparison to uninjected oocytes. The experimental data are compared with the conclusions obtained from the compilation of all available termination sequences on eukaryotic and prokaryotic mRNAs. This comparison indicates that the apparent resistance of natural termination codons against readthrough, as observed by the microinjection experiments, cannot be explained by tandem or very close second stop codons. Instead it suggests that specific context sequences around the termination codons may play a role in the efficiency of translation termination.

Animals↗
PubMed2004

Functional polypeptides can be synthesized from human mitochondrial transcripts lacking termination codons.

The human mitochondrial genome (mtDNA) is a small, circular DNA duplex found in multi-copy in the mitochondrial matrix. It is almost fully transcribed from both strands to produce large polycistronic RNA units that are processed and matured. The 13 mtDNA-encoded polypeptides are translated from mt-mRNAs that have been matured by polyadenylation of their free 3'-termini. A patient with clinical features consistent with an mtDNA disorder was recently shown to carry a microdeletion, resulting in the loss of the termination codon for MTATP6 and in its juxtaposition with MTCO3. Cell lines from this patient exhibited low steady-state levels of RNA14, the bi-cistronic transcript encoding subunits 6 and 8 of the F(o)F(1)-ATP synthase, complex V, consistent with a decreased stability. Recent reports of 'non-stop' mRNA decay systems in the cytosol have failed to determine the fate of gene products derived from transcripts lacking termination codons, although enhanced decay clearly required the 'non-stop' transcripts to be translated. We wished to determine whether functional translation products could still be expressed from non-stop transcripts in the human mitochondrion. Although a minor defect in complex V assembly was noted in the patient-derived cell lines, the steady-state level of ATPase 6 was similar to controls, consistent with the pattern of de novo mitochondrial protein synthesis. Moreover, no significant difference in ATP synthase activity could be detected. We conclude that, in the absence of a functional termination codon, although mitochondrial transcripts are more rapidly degraded, they are also translated to generate stable polypeptides that are successfully integrated into functional enzyme complexes.

Adenosine Triphosphatases↗
PubMed1995

A second, elongated, alpha 2-globin mRNA is present in reticulocytes from normal persons and subjects with terminating codon or poly A mutations.

With an RT-PCR procedure we have identified a second, elongated, alpha 2-globin mRNA in reticulocytes of normal persons and of patients with alpha-thal, particularly those with mutations in the terminating codon (TAA-->CAA; Hb Constant Spring; TAA-->TAT, Hb Paksé) or in the poly A site (AATAAA-->AATAAG). This type of mRNA is elongated because a result a cryptic poly A site 1048 bp past the terminating codon is used. Even some 5% of the alpha 2-mRNA of normal persons is of the elongated type. Quantitative data suggest high levels of this mRNA in heterozygous and homozygous carriers of poly A mutations and low levels in patients with the terminating codon mutations. Hematological and Hb data suggest that translation of these elongated mRNAs is minimal. No elongated alpha 1-mRNA has been observed.

Base Sequence↗
PubMed1983

Recognition of termination codon by release factor in the presence of a tRNA-occupied A site. Evidence for flexibility in accommodation of the release factor on the ribosome.

Recognition of the termination codon by release factor was studied with 70 S ribosomes containing initiator tRNA at the P site and an amino acid-specifying codon as well as the corresponding cognate tRNA at the A site. In vitro termination was not excluded when either the A site was occupied with an amino acid-specifying codon or the termination codon was displaced from the A site codon position by spacer nucleotides between UAA and the P site bound AUG. The release factor may have flexibility in its codon recognizing domain to be able to adjust to this enforced change in the position of the termination codon on the ribosome without loss of functional specificity. Deacylated tRNA bound at the A site of 70 S ribosomes did not interfere with stoichiometric UAA-directed release factor binding to these particles. In contrast, a ternary complex (aminoacyl-tRNA elongation factor-Tu . GTP) abolished the binding of the release factor while aminoacyl-tRNA inhibited it only weakly. This suggests that release factor and elongation factor-Tu have overlapping binding sites but the A site binding domains of the release factor and tRNA are exclusive.

Codon↗
PubMed1989

Suppression of UAA and UGA termination codons in mutant murine leukemia viruses.

Genomes of mammalian type C retroviruses contain a UAG termination codon between the gag and pol coding regions. The pol region is expressed in the form of a gag-pol fusion protein following readthrough suppression of the UAG codon. We have used oligonucleotide-directed mutagenesis to change the UAG in Moloney murine leukemia virus to UAA or UGA. These alternate termination codons were also suppressed, both in infected cells and in reticulocyte lysates. Thus, the signal or context inducing suppression of UAG in wild-type Moloney murine leukemia virus is also effective with UAA and UGA. Further, mammalian cells and cell extracts contain tRNAs capable of translating UAA and UGA as amino acids. To our knowledge, this is the first example of natural suppression of UAA in higher eucaryotes.

Animals↗
PubMed2006

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

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

Alphavirus↗
PubMed2002

An mRNA surveillance mechanism that eliminates transcripts lacking termination codons.

Translation is an important mechanism to monitor the quality of messenger RNAs (mRNAs), as exemplified by the translation-dependent recognition and degradation of transcripts harboring premature termination codons (PTCs) by the nonsense-mediated mRNA decay (NMD) pathway. We demonstrate in yeast that mRNAs lacking all termination codons are as labile as nonsense transcripts. Decay of "nonstop" transcripts in yeast requires translation but is mechanistically distinguished from NMD and the major mRNA turnover pathway that requires deadenylation, decapping, and 5'-to-3' exonucleolytic decay. These data suggest that nonstop decay is initiated when the ribosome reaches the 3' terminus of the message. We demonstrate multiple physiologic sources of nonstop transcripts and conservation of their accelerated decay in mammalian cells. This process regulates the stability and expression of mRNAs that fail to signal translational termination.

3' Untranslated Regions↗
PubMed1983

Polyamines enhance readthrough of the UGA termination codon in a mammalian messenger RNA.

The polyamines spermidine and spermine stimulate the readthrough of the UGA termination codon of rabbit beta-globin mRNA when it is translated in a rabbit reticulocyte cell-free system. The other major polyamine, putrescine, does not show this effect. The polyamine induced readthrough is specific for UGA as the UAA termination codon of alpha-globin mRNA is not read through and general translational misreading errors are not occurring in the presence of spermidine or spermine. The probable mechanism of this effect and some possible regulatory implications are discussed.

Animals↗
PubMed1999

A premature termination codon in either exon of minute virus of mice P4 promoter-generated pre-mRNA can inhibit nuclear splicing of the intervening intron in an open reading frame-dependent manner.

How premature translation termination codons (PTCs) mediate effects on nuclear RNA processing is unclear. Here we show that a PTC at nucleotide (nt) 385 in the NS1/2 shared exon of P4-generated pre-mRNAs of the autonomous parvovirus minute virus of mice caused a decrease in the accumulated levels of doubly spliced R2 relative to singly spliced R1, although the total accumulated levels of R1 plus R2 remained the same. The effect of this PTC was evident within nuclear RNA, was mediated by a PTC and not a missense transversion mutation at this position, and could be suppressed by improvement of the large intron splice sites and by mutation of the AUG that initiated translation of R1 and R2. In contrast to the PTC at nt 385, the reading frame-dependent effect of the PTC at nt 2018 depended neither on the initiating AUG nor the normal termination codon for NS2; however, it could be suppressed by a single nucleotide deletion mutation in the upstream NS1/2 common exon that shifted the 2018 PTC out of the NS2 open reading frame. This suggested that there was recognition and communication of reading frame between exons on a pre-mRNA in the nucleus prior to or concomitant with splicing.

Animals↗
PubMed1990

Identification of amino acids inserted during suppression of UAA and UGA termination codons at the gag-pol junction of Moloney murine leukemia virus.

Expression of the murine leukemia virus pol gene occurs by translational readthrough of an in-frame UAG codon between the gag and pol coding regions. In a previous study, we mutated the UAG codon to UAA or UGA and demonstrated that both of these termination codons could be suppressed in reticulocyte lysates and in infected cells with the same efficiency as UAG. We now report the identity of the amino acids inserted in vitro in response to UAA and UGA in fusion products containing the gag-pol junction region. The results show that UAA, like UAG, directs the incorporation of glutamine, whereas UGA directs the incorporation of three amino acids, arginine, cysteine, and tryptophan. To our knowledge, this is the first report indicating misreading of UAA as glutamine and UGA as arginine and cysteine in higher eukaryotes. Interestingly, although our protein synthesis system presumably contains other known UAG and UGA suppressors, these tRNAs did not suppress the termination codons in our experiments. Thus, it seems possible that the sequence surrounding the gag-pol junction not only promotes suppression but also helps determine which tRNAs function in suppression.

Amino Acid Sequence↗
PubMed2001

Congenital afibrinogenemia: mutations leading to premature termination codons in fibrinogen A alpha-chain gene are not associated with the decay of the mutant mRNAs.

Congenital afibrinogenemia is a rare coagulation disorder with autosomal recessive inheritance, characterized by the complete absence or extremely reduced levels of fibrinogen in patients' plasma and platelets. Eight afibrinogenemic probands, with very low plasma levels of immunoreactive fibrinogen were studied. Sequencing of the fibrinogen gene cluster of each proband disclosed 4 novel point mutations (1914C>G, 1193G>T, 1215delT, and 3075C>T) and 1 already reported (3192C>T). All mutations, localized within the first 4 exons of the A alpha-chain gene, were null mutations predicted to produce severely truncated A alpha-chains because of the presence of premature termination codons. Since premature termination codons are frequently known to affect the metabolism of the corresponding messenger RNAs (mRNAs), the degree of stability of each mutant mRNA was investigated. Cotransfection experiments with plasmids expressing the wild type and each of the mutant A alpha-chains, followed by RNA extraction and semiquantitative reverse-transcriptase-polymerase chain reaction analysis, demonstrated that all the identified null mutations escaped nonsense-mediated mRNA decay. Moreover, ex vivo analysis at the protein level demonstrated that the presence of each mutation was sufficient to abolish fibrinogen secretion.

Adult↗
PubMed1997

Premature termination codon mutations in the type VII collagen gene in recessive dystrophic epidermolysis bullosa result in nonsense-mediated mRNA decay and absence of functional protein.

The severe mutilating Hallopeau-Siemens type of recessive dystrophic epidermolysis bullosa (HS-RDEB) is characterized by the absence of anchoring fibrils that consist of type VII collagen. We have previously identified premature termination codon (PTC) mutations in both alleles of the type VII collagen gene (COL7A1) in HS-RDEB patients. In this study we have defined the mechanism by which these mutations elicit their phenotypic consequences in a family. The extent of nonsense-mediated mRNA decay induced by these mutations was assessed by quantitation of the level of expression of the corresponding mRNA from each of the mutant alleles by RT-PCR of parental RNA. The level of expression of the paternal mutant allele with a PTC in exon 2 was approximately 30% of that of the wild-type allele whereas that of the maternal mutant allele with a PTC in exon 104 was reduced to about 80% of the normal allele. Immunoprecipitation of newly synthesized type VII collagen with a monoclonal antibody revealed reduced quantities of alpha1(VII) polypeptides in both parents' cells, whereas their synthesis was entirely absent in the proband's keratinocytes. Thus, a consequence of these premature termination codon mutations in COL7A1 is nonsense-mediated mRNA decay, with a dramatic reduction in type VII collagen synthesis, and the absence of anchoring fibrils in the proband. These results establish a mechanistic link between the presence of premature termination codon mutations in both alleles of COL7A1 and the clinical phenotype of HS-RDEB.

Alleles↗