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PubMed2004

Combinatorial codon-based amino acid substitutions.

Twenty Fmoc-protected trinucleotide phosphoramidites representing a complete set of codons for the natural amino acids were chemically synthesized for the first time. A pool of these reagents was incorporated into oligonucleotides at substoichiometric levels to generate two libraries of variants that randomly carry either few or many codon replacements on a region encoding nine amino acids of the bacterial enzyme TEM-1 beta-lactamase. Assembly of the libraries was performed in a completely automated mode through a simple modification of ordinary protocols. This technology eliminates codon redundancy, stop codons and enables complete exploration of sequence space for single, double and triple mutations throughout a protein region spanning several residues. Sequence analysis of many non-selected clones revealed a good incorporation of the trinucleotides, producing combinations of mutations quite different from those obtained using conventional degenerate oligonucleotides. Ceftazidime-selection experiments yielded several never before reported variants containing novel amino acid combinations in the beta-lactamase omega loop region.

Amino Acid Substitution↗
PubMed2006

Four-base codon mediated mRNA display to construct peptide libraries that contain multiple nonnatural amino acids.

In vitro selection and directed evolution of peptides from mRNA display are powerful strategies to find novel peptide ligands that bind to target biomolecules. In this study, we expanded the mRNA display method to include multiple nonnatural amino acids by introducing three different four-base codons at a randomly selected single position on the mRNA. Another nonnatural amino acid may be introduced by suppressing an amber codon that may appear from a (NNK)(n) nucleotide sequence on the mRNA. The mRNA display was expressed in an Escherichia coli in vitro translation system in the presence of three types of tRNAs carrying different four-base anticodons and a tRNA carrying an amber anticodon, the tRNAs being chemically aminoacylated with different nonnatural amino acids. The complexity of the starting mRNA-displayed peptide library was estimated to be 1.1 x 10(12) molecules. The effectiveness of the four-base codon mediated mRNA display method was demonstrated in the selection of biocytin-containing peptides on streptavidin-coated beads. Moreover, a novel streptavidin-binding nonnatural peptide containing benzoylphenylalanine was obtained from the nonnatural peptide library. The nonnatural peptide library from the four-base codon mediated mRNA display provides much wider functional and structural diversity than conventional peptide libraries that are constituted from 20 naturally occurring amino acids.

Amino Acids↗
PubMed2002

Nonnatural mutagenesis in E. coli and rabbit reticulocyte lysates by using four-base codons.

Various four-base codons had been developed for introduction of nonnatural amino acids into proteins in an E. coli in vitro translation system. Here, the four-base codon mediated incorporation of nonnatural amino acids was investigated in a rabbit reticulocyte lysate. Mutated streptavidin mRNAs containing four-base codons were prepared and expressed in a rabbit reticulocyte lysate in the presence of tRNAs containing corresponding four-base anticodons. Western blot analysis indicates that various four-base codons are efficiently decoded by the corresponding tRNAs in the rabbit in vitro translation system.

Amino Acid Sequence↗
PubMed2002

Codon and rate variation models in molecular phylogeny.

This article generalizes previous models for codon substitution and rate variation in molecular phylogeny. Particular attention is paid to (1) reversibility, (2) acceptance and rejection of proposed codon changes, (3) varying rates of evolution among codon sites, and (4) the interaction of these sites in determining evolutionary rates. To accommodate spatial variation in rates, Markov random fields rather than Markov chains are introduced. Because these innovations complicate maximum likelihood estimation in phylogeny reconstruction, it is necessary to formulate new algorithms for the evaluation of the likelihood and its derivatives with respect to the underlying kinetic, acceptance, and spatial parameters. To derive the most from maximum likelihood analysis of sequence data, it is useful to compute posterior probabilities assigning residues to internal nodes and evolutionary rate classes to codon sites. It is also helpful to search through tree space in a way that respects accepted phylogenetic relationships. Our phylogeny program LINNAEUS implements algorithms realizing these goals. Readers may consult our companion article in this issue for several examples.

Algorithms↗
PubMed1996

Synonymous codon bias is related to gene length in Escherichia coli: selection for translational accuracy?

The levels of synonymous codon bias is shown to be positively correlated to gene length in Escherichia coli genes which are thought to be expressed at similar levels; these are genes whose products are present in multimeric proteins in equimolar amounts. It is argued that the positive correlation could be caused by selection to avoid missense errors during translation. Since the cost of producing a protein is proportional to its length, selection in favor of codons which increase accuracy should be greater in longer genes, and long genes should therefore have higher synonymous codon bias. It is also shown that there is variation in synonymous codon use which is independent of either expression level, gene length, amino acid composition, or chromosomal location. This variation is consistent with selection for translational accuracy but may have other origins.

Codon↗
PubMed1998

Intra- and interspecific DNA variation and codon bias of the alcohol dehydrogenase (Adh) locus in Arabis and Arabidopsis species.

Sequence variation at the alcohol dehydrogenase (Adh) locus was analyzed for six species each of the genera Arabis and Arabidopsis. Phylogenetic analysis showed that investigated species were grouped into three clusters, and the generic classification did not correspond to the clusterings. The results indicated that the genera could not be distinguished on the basis of the Adh variation. A significant difference in the ratio of silent to replacement sites was detected by MK test in two comparisons, with Arabidopsis thaliana polymorphism due to excess silent divergence. Silent changes were predominant in the evolution of the Adh locus in Arabis and Arabidopsis. To infer evolutionary significance of silent substitutions, codon bias was studied. The degree of codon bias of the Adh region was relatively constant over Arabis and Arabidopsis species. "Preferred" codons of A. thaliana were determined. No evidence of natural selection on codon change was detected in the Adh regions of A. thaliana and Arabis gemmifera.

Alcohol Dehydrogenase↗
PubMed1987

The rate of synonymous substitution in enterobacterial genes is inversely related to codon usage bias.

Genes sequences from Escherichia coli, Salmonella typhimurium, and other members of the Enterobacteriaceae show a negative correlation between the degree of synonymous-codon usage bias and the rate of nucleotide substitution at synonymous sites. In particular, very highly expressed genes have very biased codon usage and accumulate synonymous substitutions very slowly. In contrast, there is little correlation between the degree of codon bias and the rate of protein evolution. It is concluded that both the rate of synonymous substitution and the degree of codon usage bias largely reflect the intensity of selection at the translational level. Because of the high variability among genes in rates of synonymous substitution, separate molecular clocks of synonymous substitution might be required for different genes.

Biological Evolution↗
PubMed1988

"Silent" sites in Drosophila genes are not neutral: evidence of selection among synonymous codons.

The patterns of synonymous codon usage in 91 Drosophila melanogaster genes have been examined. Codon usage varies strikingly among genes. This variation is associated with differences in G+C content at silent sites, but (unlike the situation in mammalian genes) these differences are not correlated with variation in intron base composition and so are not easily explicable in terms of mutational biases. Instead, those genes with high G+C content at silent sites, resulting from a strong "preference" for a particular subset of the codons that are mostly C-ending, appear to be the more highly expressed genes. This suggests that G+C content is reduced in sequences where selective constraints are weaker, as indeed seen in a pseudogene. These and other data discussed are consistent with the effects of translational selection among synonymous codons, as seen in unicellular organisms. The existence of selective constraints on silent substitutions, which may vary in strength among genes, has implications for the use of silent molecular clocks.

Animals↗
PubMed2000

p53 codon 72 polymorphism and susceptibility to skin cancer after renal transplantation.

BACKGROUND: Infection with human papillomavirus (HPV) is an important risk factor for the development of skin cancer after renal transplantation. It has recently been suggested that degradation of the tumor suppressor gene p53 is an important mechanism for human papillomavirus-induced carcinogenesis. A common genomic polymorphism occurs at codon 72 of the p53 gene, and in vitro the codon 72Arg variant appears to be particularly susceptible to degradation. METHODS: To test the hypothesis that this polymorphism predisposes to the development of human papillomavirus-associated tumors, we studied p53 codon 72 genotype in 222 long-term survivors of renal transplantation, of whom 55 had developed at least one skin tumor. RESULTS: No differences in allele or genotype frequency were detected between individuals who had or had not developed skin tumors after transplantation, or any subgroup thereof. CONCLUSIONS: The p53 codon 72Arginine allele does not confer susceptibility to the development of skin tumors after renal transplantation.

Alleles↗
PubMed1998

HIV-1 reverse transcriptase codon 215 mutation in plasma RNA: immunologic and virologic responses to zidovudine. The AIDS Clinical Trials Group Study 175 Virology Team.

Treatment of HIV infection with zidovudine (ZDV) may select for changes in the genetic sequence of the viral reverse transcriptase (RT) that imparts drug resistance. The presence of a 2-bp mutation at codon 215 of RT (from threonine to phenylalanine or tyrosine) was assessed in plasma viral RNA in 85 subjects treated with ZDV in the AIDS Clinical Trials Group (ACTG) 175 virology substudy. Median CD4 cell numbers, HIV plasma RNA levels, and infectious titers of virus were significantly different over 56 weeks of treatment among 58 subjects with the wild-type threonine at codon 215 virus at study entry compared with the 27 subjects with mutations to phenylalanine or tyrosine (MUT) virus. Thirty percent (13 of 44 subjects) with wild-type virus at study entry developed a new codon 215 mutation. Genotypic resistance at codon 215 in plasma HIV RNA is associated with the subsequent immunologic and virologic failure of ZDV monotherapy in subjects with 200 to 500 CD4 cells/mm3.

Anti-HIV Agents↗
PubMed2005

Rare mutations at codon 103 of HIV-1 reverse transcriptase can confer resistance to non-nucleoside reverse transcriptase inhibitors.

BACKGROUND: The K103N mutation in HIV-1 reverse transcriptase (RT) confers high-level resistance to current non-nucleoside reverse transcriptase inhibitors (NNRTI). The prevalence and resistance profile of HIV-1 with other substitutions at RT codon 103 is less well documented. METHODS: K103 substitutions among over 70,000 clinical samples submitted for routine antiretroviral resistance testing at two independent centres were examined. Phenotypic resistance profiles of isolates harboring rare K103 variants in the absence of known NNRTI-associated resistance mutations were retrieved from Virco's correlative genotype/phenotype database. Genotyped samples with known treatment histories were retrieved from the British Columbia Centre for Excellence in HIV/AIDS database. Site-directed mutants containing K103 variants were constructed and phenotyped. RESULTS: K103N, R and S were observed in 29, 1.8, and 0.9% of Virco isolates and in 16, 1.5 and 0.4% of British Columbia isolates. K103T/Q/H substitutions were observed only rarely (<0.2%). The prevalence of unusual codon 103 substitutions remained stable over 5 years, except K103S, which increased over fourfold in both datasets. K103R/Q-containing clinical isolates remained phenotypically susceptible to NNRTI, whereas K103S/T/H-containing isolates showed over 10-fold decreased NNRTI susceptibility. Among patients with a known treatment history, K103S/T/H were observed primarily in individuals failing NNRTI-containing regimens. Site-directed mutants confirmed decreased susceptibility to NNRTI in K103S/T/H-containing recombinants. CONCLUSION: Variants at HIV RT codon 103 other than K103N are observed relatively rarely in clinical isolates, but K103 S, T and H confer decreased susceptibility to NNRTI. These data are relevant for interpretive genotype algorithms and in the design of assays specific to RT codon 103 mutations.

Amino Acid Substitution↗
PubMed1995

Different allelic effects of the codons 136 and 171 of the prion protein gene in sheep with natural scrapie.

Scrapie is a transmissible degenerative disease of the central nervous system occurring naturally in sheep. It belongs to the group of prion diseases also affecting man in which an abnormal isoform of the host-encoded prion protein (PrP) accumulating in the brain is responsible for neuronal death. Three main polymorphisms have been described in the sheep PrP gene, at positions 136, 154 and 171. A strong association between susceptibility/resistance to natural scrapie and a dimorphism at codon 136 of the ovine PrP gene has been reported in several breeds, including Romanov. This dimorphism, however, is not found in all scrapie-affected breeds. We have compared the PrP genotypes of Lacaune sheep obtained from enzootically affected flocks with those of apparently healthy sheep. A third variant at codon 171 was also evidenced. The results were compared with those obtained in a single experimental Romanov flock orally challenged with nematode parasites in which scrapie suddenly appeared and killed 80% of the sheep. We present evidence that, even in different epizootological circumstances, the major genetic factor controlling the susceptibility/resistance to natural scrapie in sheep, is represented by codon 171 genotype of the PrP gene. We also suggest that a modification of the allelic effects of codon 136 can occur in heavily infected animals.

Animals↗
PubMed1996

Recognition of the initiation codon for protein synthesis in foot-and-mouth disease virus RNA.

Foot-and-mouth disease virus (FMDV) RNA utilizes two in-frame initiation codons to produce two precursor proteins with identical carboxy termini. The 5' untranslated region (5'UTR) directs the ribosome to internal sequences without the need for a cap structure as used in host mRNAs. The FMDV 5'UTR was cloned upstream of the reporter gene chloramphenicol acetyltransferase (CAT) in order to study the selection of initiation site and to facilitate quantification of the translation products. After in vitro transcription with T7 RNA polymerase and translation in rabbit reticulocyte lysate, the two CAT products, resulting from initiation from the two initiation codons, were quantified. The downstream initiator AUG (AUGLb) was selected more efficiently in the wild-type 5'UTR. In truncated RNA, the upstream initiation site (AUGLab) was more efficiently utilized than in the wild-type 5'UTR. Protein synthesis initiation factors were added to translation assays to determine whether these factors influenced initiation site selection. Addition of eIF-2 and of eIF-2B changed the selection process for both types of RNA. These factors induced a 2.5-fold higher usage of the upstream AUGLab for wild-type and 5'UTR-truncated RNA. A change in mRNA concentration also induced a change in the usage of initiation codons; however, the effect of eIF-2 was measured over a broad range of mRNA concentrations. In conclusion, eIF-2 mediates the recognition of the initiation codon during both cap-dependent and internal ribosome entry site-dependent initiation.

Animals↗
PubMed2002

The roles of individual eukaryotic translation initiation factors in ribosomal scanning and initiation codon selection.

To elucidate an outline of the mechanism of eukaryotic translation initiation, 48S complex formation was analyzed on defined mRNAs in reactions reconstituted in vitro from fully purified translation components. We found that a ribosomal 40S subunit, eukaryotic initiation factor (eIF) 3, and the eIF2 ternary complex form a 43S complex that can bind to the 5'-end of an unstructured 5'-untranslated region (5'-UTR) and in the presence of eIF1 scan along it and locate the initiation codon without a requirement for adenosine triphosphate (ATP) or factors (eIF4A, eIF4B, eIF4F) associated with ATP hydrolysis. Scanning on unstructured 5'-UTRs was enhanced by ATP, eIFs 4A and 4B, and the central domain of the eIF4G subunit of eIF4F. Their omission increased the dependence of scanning on eIFs 1 and 1A. Ribosomal movement on 5'-UTRs containing even weak secondary structures required ATP and RNA helicases. eIF4F was essential for scanning, and eIFs 4A and 4B were insufficient to promote this process in the absence of eIF4F. We report that in addition to its function in scanning, eIF1 also plays a principal role in initiation codon selection. In the absence of eIF1, 43S complexes could no longer discriminate between cognate and noncognate initiation codons or sense the nucleotide context of initiation codons and were able to assemble 48S complexes on 5'-proximal AUG triplets located only 1, 2, and 4 nt from the 5'-end of mRNA.

5' Flanking Region↗
PubMed1997

GTP hydrolysis controls stringent selection of the AUG start codon during translation initiation in Saccharomyces cerevisiae.

We have isolated and characterized two suppressor genes, SUI4 and SUI5, that can initiate translation in the absence of an AUG start codon at the HIS4 locus in Saccharomyces cerevisiae. Both suppressor genes are dominant in diploid cells and lethal in haploid cells. The SUI4 suppressor gene is identical to the GCD11 gene, which encodes the gamma subunit of the eIF-2 complex and contains a mutation in the G2 motif, one of the four signature motifs that characterizes this subunit to be a G-protein. The SUI5 suppressor gene is identical to the TIF5 gene that encodes eIF-5, a translation initiation factor known to stimulate the hydrolysis of GTP bound to eIF-2 as part of the 43S preinitiation complex. Purified mutant eIF-5 is more active in stimulating GTP hydrolysis in vitro than wild-type eIF-5, suggesting that an alteration of the hydrolysis rate of GTP bound to the 43S preinitiation complex during ribosomal scanning allows translation initiation at a non-AUG codon. Purified mutant eIF-2gamma complex is defective in ternary complex formation and this defect correlates with a higher rate of dissociation from charged initiator-tRNA in the absence of GTP hydrolysis. Biochemical characterization of SUI3 suppressor alleles that encode mutant forms of the beta subunit of eIF-2 revealed that these mutant eIF-2 complexes have a higher intrinsic rate of GTP hydrolysis, which is eIF-5 independent. All of these biochemical defects result in initiation at a UUG codon at the his4 gene in yeast. These studies in light of other analyses indicate that GTP hydrolysis that leads to dissociation of eIF-2 x GDP from the initiator-tRNA in the 43S preinitiation complex serves as a checkpoint for a 3-bp codon/anticodon interaction between the AUG start codon and the initiator-tRNA during the ribosomal scanning process.

Amino Acid Sequence↗
PubMed2004

Release of initiation factors from 48S complexes during ribosomal subunit joining and the link between establishment of codon-anticodon base-pairing and hydrolysis of eIF2-bound GTP.

The 40S subunit in 48S complexes formed at the initiation codon of mRNA is bound to eukaryotic initiation factor (eIF) 3, eIF1, eIF1A, and an eIF2/GTP/Met-tRNAi(Met) ternary complex and can therefore not join a 60S subunit directly to form an 80S ribosome. We report that eIF5-induced hydrolysis of eIF2-bound GTP in 48S complexes led to release of eIF2-GDP but not eIF3 or eIF1. eIF5B did not influence factor release in the absence of 60S subunits. Therefore eIF3 and eIF1 dissociate from 40S subunits during, rather than before, the eIF5B-mediated subunit joining event. In the absence of eIF1, eIF5-stimulated hydrolysis of eIF2-bound GTP occurred at the same rate in 43S pre-initiation and 48S initiation complexes. GTP hydrolysis in 43S complexes assembled with eIF1 was much slower than in 43S or 48S complexes assembled without eIF1. Establishment of codon-anticodon base-pairing in 48S complexes relieved eIF1's inhibition. Thus, in addition to its role in initiation codon selection during 48S complex formation, eIF1 also participates in maintaining the fidelity of the initiation process at a later stage, hydrolysis of eIF2-bound GTP, by inhibiting premature GTP hydrolysis and by linking establishment of codon-anticodon base-pairing with GTP hydrolysis.

Base Pairing↗
PubMed1989

Sequences that surround the stop codons of upstream open reading frames in GCN4 mRNA determine their distinct functions in translational control.

Translational control of the GCN4 gene of Saccharomyces cerevisiae requires at least two of the four short upstream open reading frames (URFs) in the leader of GCN4 mRNA. URF4 is a strong negative element that is sufficient for repression of GCN4 expression in normal growth conditions. URF1 is approximately 30-fold less effective as a translational barrier when it is the single URF present in the mRNA leader and is required upstream from URF4 for efficient derepression of GCN4 expression under amino acid starvation conditions. We show that the last codon plus 10 bp immediately after the stop codon of URF4 are sufficient to convert URF1 into a strong translational barrier when it is present as a solitary URF. This result suggests that the characteristics of translation termination at URF4 are responsible largely for its strong inhibitory effect on translation initiation at the GCN4 AUG codon. Introduction of the same URF4 sequences at URF1 also reduces GCN4 expression under derepressing conditions when URF1 is upstream from URF4. This fact suggests that URF1 translation normally is compatible with efficient scanning and initiation downstream and that this property is required for its ability to overcome the translational barrier at URF4. These findings are consistent with the idea that ribosomes must first translate URF1 and then resume scanning in order to traverse URF4 sequences under starvation conditions. Our results indicate that nucleotides located 3' to the stop codon can be as important as those located 5' to the start site in determining the inhibitory effect of an URF on translation initiation downstream.

Base Sequence↗
PubMed2002

Shannon information theoretic computation of synonymous codon usage biases in coding regions of human and mouse genomes.

Exonic GC of human mRNA reference sequences (RefSeqs), as well as A, C, G, and T in codon position 3 are linearly correlated with genomic GC. These observations utilize information from the completed human genome sequence and a large, high-quality set of human and mouse coding sequences, and are in accord with similar determinations published by others. A Shannon Information Theoretic measure of bias in synonymous codon usage was developed. When applied to either human or mouse RefSeqs, this measure is nonlinearly correlated with genomic, exonic, and third codon position A, C, G, and T. Information values between orthologous mouse and human RefSeqs are linearly correlated: mouse = 0.092 + 0.55 human. Mouse genes were consistently placed in genomic regions whose GC content was closer to 50% than was the GC content of the human ortholog. Since the (nonlinear) information versus percent GC curve has a minimum at 50% GC and monotonically increases with increasing distance from 50% GC, this phenomenon directly results in the low slope of 0.55. This appears to be a manifestation of an evolutionary strategy for placement of genes in regions of the genome with a GC content that relates synonymous codon bias and protein folding.

Animals↗