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PubMed1991

Mutations replacing the leucine codons or altering the length of the amino acid-coding portion of the ilvGMEDA leader region of Escherichia coli.

The specificity of regulation by attenuation of the ilvGMEDA operon of Escherichia coli was examined by making alterations in the peptide-coding portion of the leader region. The effects of the alterations on attenuation control were monitored by operon fusions with the lacZ or cat gene. Substitution of the tandem leucine codons with arginine codons did not result in arginine control of attenuation even though the altered leader transcripts contained three consecutive arginine codons. Substitution of the single leucine codon with a proline codon at position 10 of the putative peptide, which had been shown to be important in the regulation of the Serratia marcescens ilv operon, did not result in control of attenuation by proline. Since the formation of neither proline nor arginine biosynthetic enzymes is regulated by attenuation control, the effect of tandem phenylalanine codons in place of the tandem leucine codons was examined and found not to result in control by phenylalanine supply. The latter failure may have been due to a configuration in the secondary structure of the protector stem of the leader transcript different from that of the wild-type transcript. The results of the study favored the idea that the lead ribosome does not initiate translation of the leader transcript until after the RNA polymerase has reached the pause site (117 bases into the leader region).

Amino Acids, Branched-Chain↗
PubMed2001

UAG readthrough in mammalian cells: effect of upstream and downstream stop codon contexts reveal different signals.

BACKGROUND: Translation termination is mediated through an interaction between the release factors eRF1 and eRF3 and the stop codon within its nucleotide context. Although it is well known that the nucleotide contexts both upstream and downstream of the stop codon, can modulate readthrough, little is known about the mechanisms involved. RESULTS: We have performed an in vivo analysis of translational readthrough in mouse cells in culture using a reporter system that allows the measurement of readthrough levels as low as 10(-4). We first quantified readthrough frequencies obtained with constructs carrying different codons (two Gln, two His and four Gly) immediately upstream of the stop codon. There was no effect of amino acid identity or codon frequency. However, an adenine in the -1 position was always associated with the highest readthrough levels while an uracil was always associated with the lowest readthrough levels. This could be due to an effect mediated either by the nucleotide itself or by the P-site tRNA. We then examined the importance of the downstream context using eight other constructs. No direct correlation between the +6 nucleotide and readthrough efficiency was observed. CONCLUSIONS: We conclude that, in mouse cells, the upstream and downstream stop codon contexts affect readthrough via different mechanisms, suggesting that complex interactions take place between the mRNA and the various components of the translation termination machinery. Comparison of our results with those previously obtained in plant cells and in yeast, strongly suggests that the mechanisms involved in stop codon recognition are conserved among eukaryotes.

Journal Article↗
PubMed2002

A cytosolic tRNA with an unmodified adenosine in the wobble position reads a codon ending with the non-complementary nucleoside cytidine.

Out of more than 500 sequenced cytosolic tRNAs, there is only one with an unmodified adenosine in the wobble position (position 34). The reason for this rare occurrence of A34 is that it is mostly deaminated to inosine-34 (I34). I34 is a common constituent in the wobble position of tRNAs and has a decoding capacity different from that of A34. We have isolated a mutant (proL207) of Salmonella typhimurium, in which the wobble nucleoside G34 has been replaced by an unmodified A in tRNA(Pro)(GGG), which is the only tRNA that normally reads the CCC codon. Thus, this mutant apparently has no tRNA that is considered cognate for the codon CCC. Despite this, the mutant grows normally. As expected, Pro-tRNA selection at the CCC codon in the A-site in a mutant deleted for the proL gene, which encodes the tRNA(Pro)(GGG), was severely reduced. However, in comparison this rate of selection was only slightly reduced in the proL207 mutant with its A34 containing tRNA(Pro)(AGG) suggesting that this tRNA reads CCC. Moreover, measurements of the interference by a tRNA residing in the P-site on the apparent termination efficiency at the A-site indicated that indeed the A34 containing tRNA reads the CCC codon. We conclude that A34 in a cytosolic tRNA is not detrimental to the cell and that the mutant tRNA(Pro)(AGG) is able to read the CCC codon like its wild-type counterpart tRNA(Pro)(GGG). We suggest that the decoding of the CCC codon by a 5'-AGG-3' anticodon occurs by a wobble base-pair between a protonated A34 and a C in the mRNA.

Adenosine↗
PubMed1998

Binary specification of nonsense codons by splicing and cytoplasmic translation.

Premature translation termination codons resulting from nonsense or frameshift mutations are common causes of genetic disorders. Complications arising from the synthesis of C-terminally truncated polypeptides can be avoided by 'nonsense-mediated decay' of the mutant mRNAs. Premature termination codons in the beta-globin mRNA cause the common recessive form of beta-thalassemia when the affected mRNA is degraded, but the more severe dominant form when the mRNA escapes nonsense-mediated decay. We demonstrate that cells distinguish a premature termination codon within the beta-globin mRNA from the physiological translation termination codon by a two-step specification mechanism. According to the binary specification model proposed here, the positions of splice junctions are first tagged during splicing in the nucleus, defining a stop codon operationally as a premature termination codon by the presence of a 3' splicing tag. In the second step, cytoplasmic translation is required to validate the 3' splicing tag for decay of the mRNA. This model explains nonsense-mediated decay on the basis of conventional molecular mechanisms and allows us to propose a common principle for nonsense-mediated decay from yeast to man.

Codon, Nonsense↗
PubMed2000

Relationship of codon bias to mRNA concentration and protein length in Saccharomyces cerevisiae.

In 1982, Ikemura reported a strikingly unequal usage of different synonymous codons, in five Saccharomyces cerevisiae nuclear genes having high protein levels. To study this trend in detail, we examined data from three independent studies that used oligonucleotide arrays or SAGE to estimate mRNA concentrations for nearly all genes in the genome. Correlation coefficients were calculated for the relationship of mRNA concentration to four commonly used measures of synonymous codon usage bias: the codon adaptation index (CAI), the codon bias index (CBI), the frequency of optimal codons (F(op)), and the effective number of codons (N(c)). mRNA concentration was best approximated as an exponential function of each of these four measures. Of the four, the CAI was the most strongly correlated with mRNA concentration (r(s)=0.62+/-0.01, n=2525, p<10(-17)). When we controlled for CAI, mRNA concentration and protein length were negatively correlated (partial r(s)=-0.23+/-0.01, n=4765, p<10(-17)). This may result from selection to reduce the size of abundant proteins to minimize transcriptional and translational costs. When we controlled for mRNA concentration, protein length and CAI were positively correlated (partial r(s)=0.16+/-0.01, n=4765, p<10(-17)). This may reflect more effective selection in longer genes against missense errors during translation. The correlation coefficients between the mRNA levels of individual genes, as measured by different investigators and methods, were low, in the range r(s)=0.39-0.68.

Codon↗
PubMed2001

Cutaneous squamous cell carcinoma and p53 codon 72 polymorphism: a need for screening?

The association between human papillomavirus (HPV)-associated cervical cancer and cutaneous squamous cell carcinoma and codon 72 polymorphism in the p53 gene is not unequivocal. Especially, it is not known whether carriers of the arginine form have an increased risk of cancer that necessitates screening. The alternative is that the polymorphism is a tumor marker instead of a risk factor. We set out a case-control study to determine the risk of squamous cell carcinoma of the skin in individuals with the p53 codon 72 arginine genotype in order to establish the possible need for screening. The distribution of the different p53 codon 72 genotypes was examined in 86 subjects with a history of cutaneous squamous cell carcinoma and in 168 controls. Additionally, 121 subjects who had had histologically proven basal cell carcinoma and 108 subjects who had had non-familial malignant melanoma were tested. p53 polymorphism was evaluated by polymerase chain reaction (PCR) using DNA samples from peripheral blood lymphocytes. In a subgroup of patients with squamous cell carcinoma and controls, the presence of epidermodyplasia verruciformis human papillomavirus (EV-HPV) DNA was determined in plucked eyebrow hair. Differences in the distributions of the genotypes among cases and controls were calculated, and univariate and multivariate analyses were performed to assess the risk to develop cutaneous squamous cell carcinoma in the presence of the p53 codon 72 arginine genotype. Frequency distributions of the three different genotypes (homozygous for the arginine allele, heterozygous for the two alleles, and homozygous for the proline allele) were similar among the squamous cell carcinoma group and the control group: 47.1%, 46.0% and 6.9% versus 47.8%, 45.8% and 6.4%, respectively. Statistical analysis showed no significant differences between these groups. In patients with squamous cell carcinoma and controls who harbored EV-HPV DNA in their plucked eyebrow hair, similar results were obtained. The distributions of the p53 codon 72 genotypes in the basal cell carcinoma and malignant melanoma group were also not significantly different from the control group. p53 codon 72 arginine homozygosity does not appear to represent a significant risk factor for cutaneous squamous cell carcinoma and screening seems not to be indicated. Mol. Carcinog. 30:56-61, 2001.

Adult↗
PubMed1999

In vitro codon-reading specificities of unmodified tRNA molecules with different anticodons on the sequence background of Escherichia coli tRNASer.

The codon-reading properties of wobble-position variants of the unmodified form of Escherichia coli tRNASer1 (the UGA anticodon) were measured in a cell-free translation system. Two variants, with the AGA and CGA anticodons, each exclusively read a single codon, UCU and UCG, respectively. The only case of efficient wobbling occurred with the variant with the GGA anticodon, which reads the UCU codon in addition to the UCC codon. Surprisingly, this wobble reading is more efficient than the Watson-Crick reading by the variant with the AGA anticodon. Furthermore, we prepared tRNA variants with AA, UC, and CU, instead of GA, in the second and third positions and measured their relative efficiencies in the reading of codons starting with UU, GA, and AG, respectively. The specificity concerning the wobble position is essentially the same as that in the case of the codons starting with UC.

Amino Acid Sequence↗
PubMed1996

High-level misincorporation of lysine for arginine at AGA codons in a fusion protein expressed in Escherichia coli.

The expression of eukaryotic genes in Escherichia coli is one of the most frequently used tools of modern science. The arginine codon AGA is a common codon in eukaryotic genes but is particularly rare in E. coli. We report here 36 to 42% misincorporation of lysine at three AGA codons in a well-expressed protein. This misincorporation yields a protein whose electrospray mass spectrum (ESMS) shows peaks at the expected mass (M), M-28, M-56 and M-84 with intensities representing 34.5(+/-0.7), 37.5(+/-1.1), 21.2(+/-1.7) and 6.6(+/-0.5) % of the total intensity, respectively. Replacement of either all three AGA codons or the two closest to the 3' end of the gene by the more common CGC arginine codon gave a protein with a single ESMS peak. Misincorporation could also be eliminated by the co-expression of the tRNA(UCL)Arg gene, argU. These studies demonstrate that misincorporation of amino acids at rare codons of recombinant proteins can be far higher than previously thought.

Amino Acid Sequence↗
PubMed1998

The modification of the wobble base of tRNAGlu modulates the translation rate of glutamic acid codons in vivo.

In Escherichia coli, uridine in the wobble position of tRNAGlu and tRNALys is modified to mnm5s2U34. This modification is believed to restrict the base-pairing capability, i.e. to prevent misreading of near-cognate codons and reduce the efficiency of cognate codon reading, especially of codons ending in G. We have determined the influence of the 5-methylaminomethyl and the 2-thio modifications of mnm5s2U34 in tRNAGlu on the translation rate of the glutamate codons GAA and GAG in vivo. In wild-type cells, GAG is translated slower (7. 7 codons/second) and GAA faster (18 codons/second) than the average codon (13 codons/second). Surprisingly, tRNAGlu lacking the 5-methylaminomethyl group, thus containing s2U34, translated GAA twofold faster (47 codons/second) and GAG fourfold slower (1.9 codons/second) than fully modified tRNAGlu. In contrast, tRNAGlu that contains mnm5U34 instead of mnm5s2U34 translated GAA fourfold slower (4.5 codons/second) and GAG only 20% slower (6.2 codons/second). Clearly, the 5-methylaminomethyl group of mnm5s2U34 facilitates base-pairing with G while decreasing base-pairing with A, resulting in rates of translation of GAG and GAA that approach that of the average codon. The 2-thio group increases the recognition of GAA and has only a minor effect on the decoding of GAG. Furthermore, the 2-thio group is important for aminoacylation (see the accompanying paper). These data imply that the function of mnm5s2U34 may be different from what has been suggested previously.

Codon↗
PubMed2000

Evolutionary change of codon usage for the histone gene family in Drosophila melanogaster and Drosophila hydei.

The nucleotide divergence in the protein-coding region for replication-dependent and replication-independent histone 3 and 4 genes of Drosophila melanogaster and Drosophila hydei occurred mostly at the synonymous site. Therefore, the pattern of codon usage was analyzed in the two species, considering the genomic codon bias, which is proposed for estimating the genomic composition pressure in the protein-coding regions. The results indicated that the codon usage in the histone gene family could be explained mostly by the genomic codon bias. However, biases for Ala and Arg were commonly observed for the histone 3 and histone 4 gene families, and biases for Ser, Leu, and Glu were observed in a gene-specific manner. This suggests that both genomic codon bias and gene- or codon-specific bias are responsible for the nucleotide differentiation in the protein-coding region of the histone genes.

Animals↗
PubMed1996

Negative effect of sequential serine codons on expression of foreign genes in Escherichia coli.

Herpes simplex virus encodes a 1298-residue protein designated ICP4 that regulates transcription of viral genes. Structural and functional analyses of ICP4 have been facilitated by production of portions of ICP4 in Escherichia coli. We previously observed that expression of most truncated forms of ICP4 in E. coli was relatively efficient, with the exception of portions of the ICP4 gene approximately between codons 160 and 220. We have now localized the portion of ICP4 that inhibits expression to a serine-rich region from position 176 to 199. Our experimental results suggest that codons within the serine-rich domain do not induce termination of transcription, do not alter the intrinsic stability of mRNA, and do not create a proteolytically sensitive site in this portion of ICP4. Silent mutations that alter codon usage of many of the 19 serine codons in this region had no effect on expression. However, we observed that the level of protein expression was inversely proportional to the number of serine codons in this region. The results are consistent with a model in which the serine-rich domain induces premature termination of translation. This effect is not due to any specific secondary structure in the mRNA or lack of sufficient seryl-tRNA synthetase. It remains to be determined whether premature termination can result from insufficient seryl-charged tRNAs. Our results suggest that foreign genes with more than 20 consecutive serine codons may be poorly expressed in E. coli.

Amino Acid Sequence↗
PubMed1993

Minor envelope proteins of duck hepatitis B virus are initiated at internal pre-S AUG codons but are not essential for infectivity.

In infected liver tissue three major and several minor duck hepatitis B virus (DHBV) envelope proteins are detectable by immunoblotting. Translation initiation at the second and the most distal internal ATG codon of the Pre-S/S gene is known to lead to synthesis of two major envelope proteins (P36 and P17) whereas the origin of a further major (P28) and other minor envelope proteins is not clear. Therefore, it was investigated whether translation initiation at pre-S ATGs leads to synthesis of these other envelope proteins and, if yes, whether they are components of viral particles and essential for infectivity. Each of the five ATG codons of the pre-S region of an infectious Chinese DHBV genome (DHBV26) was mutated separated by oligonucleotide-directed mutagenesis (point mutations or deletions) and the function of the corresponding mutant viruses were tested in vitro and in vivo. Immunoblot analysis of liver cell extracts or of extracts from hepatoma cells transfected with the DHBV genomes showed expression of minor pre-S proteins of about 35, 33, and 30 kDa. These proteins were not expressed when ATG codons at nucleotide positions 825, 882, and 957, respectively, were mutated. None of the ATG mutations abolished expression of the major P28 pre-S protein. In cell culture supernatants the minor pre-S proteins P35, P33, and P30 were identified as components of viral particles. With the exception of the DHBV genome containing the mutated ATG codon 801 (translation initiation codon for the major P36 pre-S protein) all forementioned DHBV mutant genomes were infectious. These data demonstrate that minor pre-S proteins are initiated at internal AUGs of the pre-S gene and are components of viral particles but are not essential for infectivity. In contrast to previously published speculations, the results also indicate that the major pre-S protein P28 is not initiated at AUG codon 957 but probably produced by proteolysis from larger pre-S proteins.

Animals↗
PubMed1991

Unusual codon bias occurring within insertion sequences in Escherichia coli.

The large open reading frames of insertion sequences from Escherichia coli were examined for their spatial pattern of codon usage bias and distribution of rarely used codons. There is a bias in codon usage that is generally lower toward the terminal ends of the coding regions, which is reflected in the occurrence of an excess of nonpreferred codons in the 3' portions of the coding regions as compared with the 5' portions. In contrast, typical chromosomal genes have a lower codon usage bias toward the 5' ends of the coding regions. These results imply that the selective forces reflected in codon usage bias may differ according to position within the coding sequence. In addition, these constraints apparently differ in important ways between genes contained in insertion sequences and those in the chromosome.

Chromosomes, Bacterial↗
PubMed1995

Intrastrand parity rules of DNA base composition and usage biases of synonymous codons.

When there are no biases in mutation and selection between the two strands of DNA, the 12 possible substitution rates of the four nucleotides reduces to six (type 1 parity rule or PR1), and the intrastrand average base composition is expected to be A = T and G = C at equilibrium without regard to the G + C content of DNA (type 2 parity rule or PR2). Significant deviations from the parity rules in the third codon letters of the four-codon amino acids result mostly from selective biases rather than mutational biases between the two strands of DNA during evolution. The parity rules lay the foundation for evaluating the biases in synonymous codon usage in terms of (1) directional mutation pressure for variation of the DNA G + C content due to mutational biases between alpha-bases (A or T) and gamma-bases (G or C), (2) strand-bias mutation, for example, by DNA repair during transcription, and (3) functional selection in evolution, for example, due to tRNA abundance. The present analysis shows that, although the PR2 violation is common in the third codon letters of four-codon amino acids, the contribution of PR2 violation to the DNA G + C content of the third codon position is small and, in majority of cases, mildly counteracts the effect of the directional mutation pressure on the G + C content.

Animals↗
PubMed1995

On codon reassignment.

Schultz and Yarus (J. Mol. Biol. 235:1377-1380, 1994) have proposed that reassignment of codons in the genetic code passes through a stage in which the codons are ambiguously translated. In contrast we state that such ambiguity would be deleterious, and that, to be reassigned, a codon, together with the tRNA that translates the codon, must first disappear from coding sequences, after which a tRNA appears with a mutated anticodon, and this enables the codon to reappear with a changed meaning. In the case of a stop codon, the relevant release factor must change so as to recognize it.

Codon↗
PubMed1991

Codon usage in Aspergillus nidulans.

Synonymous codon usage in genes from the ascomycete (filamentous) fungus Aspergillus nidulans has been investigated. A total of 45 gene sequences has been analysed. Multivariate statistical analysis has been used to identify a single major trend among genes. At one end of this trend are lowly expressed genes, whereas at the other extreme lie genes known or expected to be highly expressed. The major trend is from nearly random codon usage (in the lowly expressed genes) to codon usage that is highly biased towards a set of 19-20 "optimal" codons. The G + C content of the A. nidulans genome is close to 50%, indicating little overall mutational bias, and so the codon usage of lowly expressed genes is as expected in the absence of selection pressure at silent sites. Most of the optimal codons are C- or G- ending, making highly expressed genes more G + C-rich at silent sites.

Aspergillus nidulans↗
PubMed1989

Context specific misreading of phenylalanine codons.

It has previously been shown that the phenylalanine codon UUC encoding residue 8 of the Escherichia coli argI gene product, ornithine transcarbamylase, is misread as leucine at a high frequency during phenylalanine starvation. However, no misreading of the UUU encoding residue 3 was observed under these conditions. Using oligonucleotide-directed, site-specific mutagenesis, we have constructed mutants where these codons have been changed. Using these mutant argI genes we see a high level of mistranslation at position 8 during phenylalanine starvation whether the codon is UUU or UUC. With either codon at position 3 we see no leucine substitution. We also constructed a gene with a leucine codon at position 3. The product of this latter mutated gene is stable and active, indicating that preferential turnover of mistranslated protein is not obscuring an otherwise high rate of misreading. This would seem to indicate that it is the context rather than the particular phenylalanine codon which is important in determining these misreading levels.

Amino Acid Sequence↗
PubMed1988

Release factor competition is equivalent at strong and weakly suppressed nonsense codons.

We have compared the competition between strong or weak suppressor tRNAs and translational release factors (RF) at nonsense codons in the lacI gene of Escherichia coli. Using the F'lacIZ fusions developed by Miller and coworkers, UAG, UAA, and UGA codons at positions 189 and 220 were efficiently suppressed by plasmid-borne tRNA(trp) suppressors cognate to each nonsense triplet. Introduction of a compatible RF 1 plasmid competed at UAG and UAA but not UGA codons. An RF2 expressing plasmid competed at UAA and UGA but had little effect at UAG. Release factor competition against weak suppressors was measured using combinations of noncognate suppressors and nonsense codons. In each case, release factor plasmids behaved identically towards poorly suppressed codons as they did when the same codons were efficiently suppressed. The implications for these studies on the role of release factors in nonsense suppression context effects are discussed.

Codon↗