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PubMed1998

The association of nonsense codons with exon skipping.

Some genes that contain premature nonsense codons express alternatively-spliced mRNA that has skipped the exon containing the nonsense codon. This paradoxical association of translation signals (nonsense codons) and RNA splicing has inspired numerous explanations. The first is based on the fact that premature nonsense codons often reduce mRNA abundance. The reduction in abundance of full-length mRNA then allows more efficient amplification during PCR of normal, minor, exon-deleted products. This mechanism has been demonstrated to explain an extensive correlation between nonsense codons and exon-skipping for the hamster Hprt gene. The second explanation is that the mutation producing an in-frame nonsense codon has an effect on exon definition. This has been demonstrated for the Mup and hamster Hprt gene by virtue of the fact that missense mutations at the same sites also are associated with the same exon-deleted mRNA. The third general explanation is that a hypothetical process takes place in the nucleus that recognizes nonsense codons, termed 'nuclear scanning', which then has an effect on mRNA splicing. Definitive evidence for nuclear scanning is lacking. My analysis of both nonsense and missense mutations associated with exon skipping in a large number of genes revealed that both types of mutations frequently introduce a T into a purine-rich DNA sequence and are often within 30 base pairs of the nearest exon boundary. This is intriguing given that purine-rich splicing enhancers are known to be inhibited by the introduction of a T. Almost all mutations associated with exon skipping occur in purine-rich or A/C-rich sequences, also characteristics of splicing enhancers. I conclude that most cases of exon skipping associated with premature termination codons may be adequately explained either by a structural effect on exon definition or by nonquantitative methods to measure mRNA, rather than an effect on a putative nuclear scanning mechanism.

Animals↗

PubMed1996

Premature nonsense codons decrease the stability of phytohemagglutinin mRNA in a position-dependent manner.

Premature termination of translation has often been associated with decreased mRNA accumulation in plants, but the affected step in gene expression has not been identified. To investigate this problem, the expression of wild-type and mutant alleles of the bean phytohemagglutinin (PHA) gene has been examined in tobacco cells and transgenic plants. Measurement of mRNA decay rates in stably transformed cell lines demonstrated that premature nonsense codons markedly destabilized the mRNA. This decreased stability was also reflected by decreased accumulation of transcripts containing premature nonsense codons in transgenic plants. The positional dependence of the nonsense codon effect was evaluated by introducing premature nonsense codons at different distances from the PHA AUG start codon. Transcripts with nonsense codons about 20, 40 or 60% of the way through the normal PHA coding region yielded highly unstable mRNAs, whereas a transcript with a nonsense codon at 80% was as stable as wild-type. The ability to recognize and rapidly degrade certain transcripts with early nonsense codons could provide plant cells with a means to minimize the production of wasteful and possible deleterious truncated proteins.

Alleles↗

PubMed2002

Polypeptide release at sense and noncognate stop codons by localized charge-exchange alterations in translational release factors.

The mechanism of stop codon recognition during translation has long been a puzzle. Only recently has it been established that a tripeptide in the bacterial release factors (RFs) 1 and 2 serves as the "anticodon" in deciphering stop codons in mRNA. However, the molecular basis of the accuracy of stop codon recognition is unknown. Although specific tripeptides in the RFs are primarily responsible for selective reading of cognate stop codons, charge-flip variant RF proteins, altered at conserved Glu residues adjacent to the tripeptide-anticodon, are shown here to be crucial to codon recognition. Changes of these Glu residues are capable of triggering polypeptide release at noncognate stop codons and also at sense codons. These changes also reverse the growth inhibition by RFs containing "harmful" tripeptide-anticodon changes. These findings suggest that electrostatic interactions involving negative charges in domain C of the RFs mediate their accurate docking in the ribosome. Our results also establish that the charge flipping creates a phenotype/translation termination by "codon bypassing" via relaxed positioning of the RF tripeptide-anticodon in the decoding pocket of the ribosome.

Amino Acid Sequence↗

PubMed2026

Codon bias variation in Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus causes a multiplicity of human diseases acquired in community and healthcare settings alike around the globe. While most studies focus on coding changes to assess genome evolution and study genetic adaptation, interrogation of silent mutations in the form of synonymous codon usage bias is less well-studied. As such, understanding of patterns in codon bias at the gene and genome levels, and how codon bias impacts protein expression in S. aureus remains incomplete. METHODS: The codon bias of 2,565 protein encoding genes from NCTC 8325 was queried against all publicly available closed S. aureus genomes. Using public BioSample data, genomes were sorted by disease state, submitting institution, and collection site. Codon bias was assessed at the level of gene and genome using the codon adaptation index (CAI), calculated using 30S and 50S ribosomal genes. Gene set enrichment analysis was applied to determine associations between physiological functions, CAI gene scores, and interquartile ranges. CAI scores were also compared to an in vitro S. aureus proteomics database to correlate codon bias and protein expression. RESULTS: CAI scores varied within and between isolates at the gene and genome levels. Genes with ribosome-associated functions were most enriched among high CAI genes, and had low CAI interquartile ranges (IQR), suggesting selective pressure to maintain high expression of these genes across all S. aureus isolates. Genome sequences submitted by Aga Khan University Hospital, Nairobi, Kenya were most different from others. For the LAC USA 300 strain, CAI and protein expression were moderately positively correlated (cor = 0.534, p < 2.2e-16). CONCLUSIONS: Codon bias in S. aureus was shown to vary between gene, and to be a source of genetic variation between isolates; CAI and in vitro protein expression were positively correlated.

Staphylococcus aureus↗

PubMed1995

Ultraviolet-B-light-induced mutagenesis of C-H-ras codons 11 and 12 in human skin fibroblasts.

Mutations in the ras oncogene are detected with a high frequency in non-melanoma skin cancer. Approximately half of the squamous-cell carcinomas (SCC) and one third of the basal-cell carcinomas (BCC) carry mutations at the second position of Ha-ras codon 12 (GGC to GTC), whereas mutations in Ki-ras codon 12 occur less frequently. Since the mutations in the Ha-ras and Ki-ras oncogenes are located opposite potential pyrimidine dimer sites (C-C), it is likely that the mutations are induced by ultraviolet radiation present in sunlight. We studied the capacity of ultraviolet B (UVB) light to induce base-pair changes in Ha-ras codons 11 and 12 in human skin fibroblasts. UVB induced mostly C to T and G to A transitions and C to A and G to T transversions. The base-pair change with the highest relative abundance was C to T in the middle position of codon 11 followed by (in diminishing relative abundance) C to A in the middle position of codon 11, G to A and G to T in the middle position of codon 12. The C to T and G to A transitions are compatible with pyrimidine photodimers as pre-mutagenic lesions, whereas the C to A and G to T transversions could be generated due to the formation of 8-hydroxyguanine, which is the major oxidation product of guanine. The relative abundance of mutations induced by UVB in Ha-ras codons 11 and 12 does not correlate with mutations observed in the DNA from non-melanoma skin cancer, where the G to T transversion in the middle position of codon 12 is selected.

Base Sequence↗

PubMed1994

Ultraviolet B light-induced mutagenesis of p53 hotspot codons 248 and 249 in human skin fibroblasts.

Mutations in the p53 tumor suppressor gene are detected in approximately half of non-melanoma skin cancers. The type of base-pair changes observed strongly suggests solar radiation as the causative mutagen. Mutations are distributed nonrandomly and form moderate hotspots. We studied the capacity of ultraviolet B light (UVB, 280-320 nm) to induce base-pair changes into the p53 exon 7 sequence extending from nt 14067 to 14075 in human skin fibroblasts. This sequence contains hotspot codon 248. UVB induced mostly C-->A and G-->T transversions. The base-pair change with the highest relative abundance was C-->A in the first position of codon 250 (CCC-->ACC), followed by (in diminishing relative abundance) G-->T in the third position of codon 249 (AGG-->AGT), C-->A in the first position of codon 248 (CGG-->AGG), and C-->A in the third position of codon 247 (AAC-->AAA). The C-->T transition in the third position of codon 247 (AAC-->AAT) occurred with moderate efficiency. These base-pair changes are compatible with pyrimidine photodimers as premutagenic lesions, but they could also form opposite 8-hydroxyguanine, which is the major oxidation product of guanine. No evidence was obtained for the presence of tandem double CC-->TT transitions in the untranscribed strand at codons 247/248 and 250. The relative abundance of mutations induced by UVB in the p53 sequence extending from codon 247 to 250 in human fibroblasts does not correlate with mutations observed in the DNA from non-melanoma skin cancer. This lack of correlation suggests that the mutability of this p53 sequence at the DNA level plays only a minor role in the pathogenesis of non-melanoma skin cancer in humans.

Base Sequence↗

PubMed1995

Benzo[a]pyrene-induced mutagenesis of p53 hot-spot codons 248 and 249 in human hepatocytes.

Human tobacco-related cancers show a high frequency of G-to-T transversions in several mutation hot-spot regions of the p53 tumor suppressor gene, probably the result of specific mutagens in tobacco smoke, most notably benzo[a]pyrene. To gain insight into the mechanism of formation of these G-to-T transversions in tobacco-associated carcinogenesis, we studied the mutagenesis of p53 codons 247-250 by benzo[a]pyrene in human hepatocellular carcinoma cells by restriction fragment length polymorphism-polymerase chain reaction genotypic analysis. Benzo[a]pyrene preferentially induced G-to-T transversion in the second and third positions of codon 248 and C-to-A transversion in the first position of codon 248. However, benzo[a]pyrene did not induce base-pair changes in codon 249, which is a mutational hot-spot in aflatoxin-related hepatocarcinogenesis, in which predominantly G-to-T transversion in the third position of codon 249 is observed. The benzo[a]pyrene-induced G-to-T transversion in the middle position of codon 248, in which arginine is changed into leucine, is frequently observed in tumors of the lung. The other two benzo[a]pyrene-induced base-pair changes in codon 248, namely the C-to-A transversion in the first position and G-to-T transversion in the third position, do not lead to a change in the amino-acid composition of the p53 protein. These mutations are silent and therefore are not selected in tumors. It follows that benzo[a]pyrene-induced mutability on the DNA level in p53 codons 247-250 correlates well with the type of mutation found in tumors of the lung. Therefore, our results support the hypothesis that benzo[a]pyrene is the etiological agent in tobacco-related cancers.

Base Composition↗

PubMed1993

Synonymous codon usage in Kluyveromyces lactis.

The nature and variation of synonymous codon usage in 47 open reading frames from Kluyveromyces lactis have been investigated. Using multivariate statistical analysis, a single major trend among K. lactis genes was identified that differentiates among genes by expression level: highly expressed genes have high codon usage bias, while genes of low expression level have low bias. A relatively minor secondary trend differentiates among genes according to G+C content at silent sites. In these respects, K. lactis is similar to both Saccharomyces cerevisiae and Candida albicans, and the same 'optimal' codons appear to be selected in highly expressed genes in all three species. In addition, silent sites in K. lactis and S. cerevisiae have similar G+C contents, but in C. albicans genes they are more A+T-rich. Thus, in all essential features, codon usage in K. lactis is very similar to that in S. cerevisiae, even though silent sites in genes compared between these two species have undergone sufficient mutation to be saturated with changes. We conclude that the factors influencing overall codon usage, namely mutational biases and the abundances of particular tRNAs, have not diverged between the two species. Nevertheless, in a few cases, codon usage differs between homologous genes from K. lactis and S. cerevisiae. The strength of codon usage bias in cytochrome c genes differs considerably, presumably because of different expression patterns in the two species. Two other, linked, genes have very different G+C content at silent sites in the two species, which may be a reflection of their chromosomal locations. Correspondence analysis was used to identify two open reading frames with highly atypical codon usage that are probably not genes.

Base Sequence↗

PubMed1999

Cloning and characterization of the gene encoding the highly expressed ribosomal protein l3 of the ciliated protozoan Tetrahymena thermophila. Evidence for differential codon usage in highly expressed genes.

We have cloned and characterized the cDNA and the macronuclear genomic copy of the highly conserved ribosomal protein (r-protein) L3 of Tetrahymena thermophila. The r-protein L3 is encoded by a single copy gene interrupted by one intron. The organization of the promoter region exhibits features characteristic of ribosomal protein genes in Tetrahymena. The codon usage of the L3 gene is highly biased. A thorough analysis of codon usage in Tetrahymena genes revealed that genes could be categorized into two classes according to codon usage bias. Class A comprises r-protein genes and a number of other highly expressed genes. Class B comprises weakly expressed genes such as the conjugation induced CnjB and CnjC genes, but surprisingly, this class also contains abundantly expressed genes such as the genes encoding the surface antigens SerH3 and SerH1. Codon usage is slightly more restricted in class A than in class B, but both classes exhibit distinct and different codon usage biases. Class A genes preferentially use C and U in the silent third codon positions, whereas class B genes preferentially use A and U in the silent third codon positions. The analysis suggests that two different strategies have been employed for optimization of codon usage in the A+T-rich genome of Tetrahymena.

Animals↗

PubMed1996

On the mechanism of leftward frameshifting at several hungry codons.

We have used lacZ reporter genes to assess leftward ribosome frameshifting on sequences containing the quadruplet U UUC followed by several different triplets coding for lysine, isoleucine, or leucine. Limitation for lysine-tRNA provokes leftward frameshifting when the slippery quadruplet is followed by either lysine codon aag or aaa, but not when followed by an isoleucine or leucine codon. Limitation for isoleucine provokes frameshifting when the quadruplet is followed by either isoleucine codon aua or auc, but not when it is followed by a lysine codon. We conclude that the quadruplet promotes shifting when the ribosome is stalled at any "hungry" codon immediately after it. Changing the quadruplet to U AGC, at which peptidyl-tRNA cognate to the AGC triplet will be mismatched at all three anticodon positions if it slips left, abolishes frameshifting when the ribosome is stalled at the next position. We conclude that the U UUC quadruplet promotes frameshifting by virtue of its ability to pair with a left-slipped peptidyl-tRNA. The frameshift promoted by isoleucine-tRNA limitation of the U UUC aua sequence was analyzed by amino acid sequencing of the protein product. It occurs through reading of the Cau histidine codon overlapping the hungry codon from the left. This result rules out a "simultaneous slippage" type of mechanism. It strongly suggests instead that starvation-promoted frameshifting occurs primarily by slippage of peptidyl-tRNA just upstream of the stall site, followed by decoding of the triplet overlapping the stall site from the left or 5' side. A secondary finding is that the last base of the "hungry" codon has a moderate effect on its shiftiness, aag being shiftier than aaa, and aua being shiftier than auc.

Amino Acid Sequence↗

PubMed1996

Specific replacement of consecutive AGG codons results in high-level expression of human cardiac troponin T in Escherichia coli.

The adult isoform of human cardiac troponin T (TnT) contains 288 amino acids, 14 of which (4.9%) are encoded by the rarely used arginine codons (12 AGG, 2 AGA) in Escherichia coli genes. To generate sufficient quantity of TnT protein for antibody production, we cloned the corresponding cDNA and expressed it in E. coli. A low-level expression of TnT that comprised only about 1% of total cell protein was initially observed with the use of the native cDNA. The existence of two pairs of consecutive AGG codons AGG(165) AGG(166) and AGG(215) AGG(216) in the cDNA was suspected to be the main cause for this low-level expression. These two pairs of consecutive AGG codons were successively replaced with the major synonymous codon CGT by site-directed mutagenesis. As suspected, a 10-fold increase in TnT expression was obtained when one pair of the rare arginine codons was replaced and a 40-fold increase was achieved when both pairs of the rare codons were replaced. Our finding demonstrates the importance of consecutive rare codons in the suppression of high-level expression of heterologous proteins in E. coli and suggests that in order to maximize protein expression, a similar approach may be taken with other genes which contain consecutive rare codons.

Adult↗

PubMed1999

Involvement of the aphthovirus RNA region located between the two functional AUGs in start codon selection.

Initiation of translation in picornavirus RNAs occurs internally, mediated by an element termed internal ribosome entry site (IRES). In the aphthovirus RNA, the IRES element directs translation initiation at two in-frame AUGs separated by 84 nucleotides. We have found that bicistronic constructs that contained the IRES element followed by the fragment including the aphthovirus start codons in front of the second gene mimicked the translation initiation pattern of viral RNA observed in infected cells. In those constructs, the frequency of initiation at the first AUG was increased by a sequence context that resembled the favorable consensus for cap-dependent translation, although initiation at the second site was always preferred. In addition, we have found that initiation at the second start codon was not diminished under conditions in which the first initiation codon was blocked by antisense oligonucleotide interference. Interestingly, mutations that positioned the second AUG out-of-frame with the first AUG did not interfere with the frequency of initiation at the second one. On the contrary, IRES-dependent translation initiation in bicistronic constructs lacking the sequences present between functional AUGs in the viral RNA was sensitive to the presence of out-of-frame initiator codons and hairpins in the spacer region. This remarkable difference in start codon recognition was due to the nucleotide composition of the RNA that separated the IRES from the initiator codon. Thus our results indicate that the region located in the aphthovirus RNA between functional AUGs is involved in start codon recognition, strongly favoring selection of the second start AUG as the main initiator codon.

Animals↗

PubMed1986

An evolutionary perspective on synonymous codon usage in unicellular organisms.

Observed patterns of synonymous codon usage are explained in terms of the joint effects of mutation, selection, and random drift. Examination of the codon usage in 165 Escherichia coli genes reveals a consistent trend of increasing bias with increasing gene expression level. Selection on codon usage appears to be unidirectional, so that the pattern seen in lowly expressed genes is best explained in terms of an absence of strong selection. A measure of directional synonymous-codon usage bias, the Codon Adaptation Index, has been developed. In enterobacteria, rates of synonymous substitution are seen to vary greatly among genes, and genes with a high codon bias evolve more slowly. A theoretical study shows that the patterns of extreme codon bias observed for some E. coli (and yeast) genes can be generated by rather small selective differences. The relative plausibilities of various theoretical models for explaining nonrandom codon usage are discussed.

Amino Acid Sequence↗

PubMedDate not supplied

Selection pressures on codon usage in the complete genome of bacteriophage T7.

We searched the complete 39,936 base DNA sequence of bacteriophage T7 for nonrandomness that might be attributed to natural selection. Codon usage in the 50 genes of T7 is nonrandom, both over the whole code and among groups of synonymous codons. There is a great excess of purine- any base-pyrimidine (RNY) codons. Codon usage varies between genes, but from the pooled data for the whole genome (12,145 codons) certain putative selective constraints can be identified. Codon usage appears to be influenced by host tRNA abundance (particularly in highly expressed genes), tRNA-mRNA (one such interaction being perhaps responsible for maintaining the excess of RNY codons) and a lack of short palindromes. This last constraint is probably due to selection against host restriction enzyme recognition sites; this is the first report of an effect of this kind on codon usage. Selection against susceptibility to mutational damage does not appear to have been involved.

Base Sequence↗

PubMed1991

Correlations between the compositional properties of human genes, codon usage, and amino acid composition of proteins.

We have analyzed the correlation that exists between the GC levels of third and first or second codon position for about 1400 human coding sequences. The linear relationship that was found indicates that the large differences in GC level of third codon positions of human genes are paralleled by smaller differences in GC levels of first and second codon positions. Whereas third codon position differences correspond to very large differences in codon usage within the human genome, the first and second codon position differences correspond to smaller, yet very remarkable, differences in the amino acid composition of encoded proteins. Because GC levels of codon positions are linearly correlated with the GC levels of the isochores harboring the corresponding genes, both codon usage and amino acid composition are different for proteins encoded by genes located in isochores of different GC levels. Furthermore, we have also shown that a linear relationship with a unit slope and a correlation coefficient of 0.77 exists between GC levels of introns and exons from the 238 human genes currently available for this analysis. Introns are, however, about 5% lower in GC, on average, than exons from the same genes.

Amino Acid Sequence↗

PubMed1998

Selection on the codon bias of chloroplast and cyanelle genes in different plant and algal lineages.

In the plant chloroplast genome the codon usage of the highly expressed psbA gene is unique and is adapted to the tRNA population, probably due to selection for translation efficiency. In this study the role of selection on codon usage in each of the fully sequenced chloroplast genomes, in addition to Chlamydomonas reinhardtii, is investigated by measuring adaptation to this pattern of codon usage. A method is developed which tests selection on each gene individually by constructing sequences with the same amino acid composition as the gene and randomly assigning codons based on the nucleotide composition of noncoding regions of that genome. The codon bias of the actual gene is then compared to a distribution of random sequences. The data indicate that within the algae selection is strong in Cyanophora paradoxa, affecting a majority of genes, of intermediate intensity in Odontella sinensis, and weaker in Porphyra purpurea and Euglena gracilis. In the plants, selection is found to be quite weak in Pinus thunbergii and the angiosperms but there is evidence that an intermediate level of selection exists in the liverwort Marchantia polymorpha. The role of selection is then further investigated in two comparative studies. It is shown that average relative codon bias is correlated with expression level and that, despite saturation levels of substitution, there is a strong correlation among the algae genomes in the degree of codon bias of homologous genes. All of these data indicate that selection for translation efficiency plays a significant role in determining the codon bias of chloroplast genes but that it acts with different intensities in different lineages. In general it is stronger in the algae than the higher plants, but within the algae Euglena is found to have several unusual features which are noted. The factors that might be responsible for this variation in intensity among the various genomes are discussed.

Chloroplasts↗

PubMed1998

An evaluation of measures of synonymous codon usage bias.

Synonymous codons are not generally used at equal frequencies, and this trend is observed for most genes and organisms. Several methods have been proposed and used to estimate the degree of the nonrandom use of the different synonymous codons. The estimates obtained by these methods, however, show different levels of both precision and dispersion when coding regions of a finite number of codons are under analysis. Here, we present a study, based on computer simulation, of how the different methods proposed to evaluate the nonrandom use of synonymous codons are affected by the length of the coding region analyzed. The results show that some of these methods are heavily influenced by the number of codons and that the comparison of codon usage bias between coding regions of different lengths shows a methodological bias under different conditions of nonrandom use of synonymous codons. The study of the dispersion of the estimates obtained by the different methods gives, on the other hand, an indication of the methods to be applied to compare values of codon usage bias among coding regions of equivalent length.

Animals↗

PubMed2000

In vivo analysis of mutated initiation codons in the mitochondrial COX2 gene of Saccharomyces cerevisiae fused to the reporter gene ARG8m reveals lack of downstream reinitiation.

To examine normal and aberrant translation initiation in Saccharomyces cerevisiae mitochondria, we fused the synthetic mitochondrial reporter gene ARG8m to codon 91 of the COX2 coding sequence and inserted the chimeric gene into mitochondrial DNA (mtDNA). Translation of the cox2(1-91)::ARG8m mRNA yielded a fusion protein precursor that was processed to yield wild-type Arg8p. Thus mitochondrial translation could be monitored by the ability of mutant chimeric genes to complement a nuclear arg8 mutation. As expected, translation of the cox2(1-91)::ARG8m mRNA was dependent on the COX2 mRNA-specific activator PET111. We tested the ability of six triplets to function as initiation codons in both the cox2(1-91)::ARG8m reporter mRNA and the otherwise wild-type COX2 mRNA. Substitution of AUC, CCC or AAA for the initiation codon abolished detectable translation of both mRNAs, even when PET111 activity was increased. The failure of these mutant cox2(1-91)::ARG8m genes to yield Arg8p demonstrates that initiation at downstream AUG codons, such as COX2 codon 14, does not occur even when normal initiation is blocked. Three mutant triplets at the site of the initiation codon supported detectable translation, with efficiencies decreasing in the order GUG, AUU, AUA. Increased PET111 activity enhanced initiation at AUU and AUA codons. Comparisons of expression, at the level of accumulated product, of cox2(1-91)::ARG8m and COX2 carrying these mutant initiation codons revealed that very low-efficiency translation can provide enough Cox2p to sustain significant respiratory growth, presumably because Cox2p is efficiently assembled into stable cytochrome oxidase complexes.

Amino Acid Sequence↗