First position wobble in codon-anticodon pairing: amber suppression by a yeast glutamine tRNA

A 2.4-kb fragment of DNA isolated from the Saccharomyces cerevisiae genome was found to suppress amber mutations when its carrier plasmid was present in high copy number. A 1.2-kb subclone of this fragment was sufficient to confer suppressor activity. Sequencing has established that this fragment carries a normal glutamine tRNA gene. Deletion of this tRNA gene from the subclone resulted in the loss of suppressor activity. The tRNAGln has the anticodon CUG that normally recognizes the glutamine codon CAG. We propose that suppression occurs via an inefficient readthrough of the UAG amber stop codons during translation. Such readthrough requires wobble in the first position of the codon.     

Coevolution of codon usage and tRNA genes leads to alternative stable states of biased codon usage

The typical number of tRNA genes in bacterial genomes is around 50, but this number varies from under 30 to over 120. We argue that tRNA gene copy numbers evolve in response to translational selection. In rapidly multiplying organisms, the time spent in translation is a limiting factor in cell division; hence, it pays to duplicate tRNA genes, thereby increasing the concentration of tRNA molecules in the cell and speeding up translation. In slowly multiplying organisms, translation time is not a limiting factor, so the overall translational cost is minimized by reducing the tRNAs to only one copy of each required gene. Translational selection also causes a preference for codons that are most rapidly translated by the current tRNAs; hence, codon usage and tRNA gene content will coevolve to a state where each is adapted to the other. We show that there is often more than one stable coevolved state. This explains why different combinations of tRNAs and codon bias can exist for different amino acids in the same organism. We analyze a set of 80 complete bacterial genomes and show that the theory predicts many of the trends that are seen in these data.     

Patterns of context-dependent codon biases

The association of codon context and codon usage was studied in seven bacteria as well as Schizosaccharomyces pombe and Encephalitozoon cuniculi. The association is strongest in magnitude closest to the codons of interest but there is apparently no rule about which of the two contexts is generally strongest associated to codon usage. In all bacterial species and in the intron-rich Sch. pombe it was furthermore observed from plots of chi2 versus N that the wobble positions of codons in the proximity cause regular peaks both upstream and downstream. This observation is discussed in relation to a possible effect of mutational pressure on the association of codon usage and codon context. Absence of peaks corresponding to the wobble positions in the intron-poor En. cuniculi, and presence in Sch. pombe, may indicate that the role of introns in the context-dependent codon bias is negligible.     

The RNA polymerase III-dependent family of genes in hemiascomycetes: comparative RNomics, decoding strategies, transcription and evolutionary implications

We present the first comprehensive analysis of RNA polymerase III (Pol III) transcribed genes in ten yeast genomes. This set includes all tRNA genes (tDNA) and genes coding for SNR6 (U6), SNR52, SCR1 and RPR1 RNA in the nine hemiascomycetes Saccharomyces cerevisiae, Saccharomyces castellii, Candida glabrata, Kluyveromyces waltii, Kluyveromyces lactis, Eremothecium gossypii, Debaryomyces hansenii, Candida albicans, Yarrowia lipolytica and the archiascomycete Schizosaccharomyces pombe. We systematically analysed sequence specificities of tRNA genes, polymorphism, variability of introns, gene redundancy and gene clustering. Analysis of decoding strategies showed that yeasts close to S.cerevisiae use bacterial decoding rules to read the Leu CUN and Arg CGN codons, in contrast to all other known Eukaryotes. In D.hansenii and C.albicans, we identified a novel tDNA-Leu (AAG), reading the Leu CUU/CUC/CUA codons with an unusual G at position 32. A systematic 'p-distance tree' using the 60 variable positions of the tRNA molecule revealed that most tDNAs cluster into amino acid-specific sub-trees, suggesting that, within hemiascomycetes, orthologous tDNAs are more closely related than paralogs. We finally determined the bipartite A- and B-box sequences recognized by TFIIIC. These minimal sequences are nearly conserved throughout hemiascomycetes and were satisfactorily retrieved at appropriate locations in other Pol III genes.     

tRNA gene copy number variation in humans

The human tRNAome consists of more than 500 interspersed tRNA genes comprising 51 anticodon families of largely unequal copy number. We examined tRNA gene copy number variation (tgCNV) in six individuals; two kindreds of two parents and a child, using high coverage whole genome sequence data. Such differences may be important because translation of some mRNAs is sensitive to the relative amounts of tRNAs and because tRNA competition determines translational efficiency vs. fidelity and production of native vs. misfolded proteins. We identified several tRNA gene clusters with CNV, which in some cases were part of larger iterations. In addition there was an isolated tRNALysCUU gene that was absent as a homozygous deletion in one of the parents. When assessed by semiquantitative PCR in 98 DNA samples representing a wide variety of ethnicities, this allele was found deleted in hetero- or homozygosity in all groups at ~ 50% frequency. This is the first report of copy number variation of human tRNA genes. We conclude that tgCNV exists at significant levels among individual humans and discuss the results in terms of genetic diversity and prior genome wide association studies (GWAS) that suggest the importance of the ratio of tRNALys isoacceptors in Type-2 diabetes.     

Transfer RNA structural change is a key element in the reassignment of the CUG codon in Candida albicans.

The human pathogenic yeast Candida albicans and a number of other Candida species translate the standard leucine CUG codon as serine. This is the latest addition to an increasing number of alterations to the standard genetic code which invalidate the theory that the code is frozen and universal. The unexpected finding that some organisms evolved alternative genetic codes raises two important questions: how have these alternative codes evolved and what evolutionary advantages could they create to allow for their selection? To address these questions in the context of serine CUG translation in C.albicans, we have searched for unique structural features in seryl-tRNA(CAG), which translates the leucine CUG codon as serine, and attempted to reconstruct the early stages of this genetic code switch in the closely related yeast species Saccharomyces cerevisiae. We show that a purine at position 33 (G33) in the C.albicans Ser-tRNA(CAG) anticodon loop, which replaces a conserved pyrimidine found in all other tRNAs, is a key structural element in the reassignment of the CUG codon from leucine to serine in that it decreases the decoding efficiency of the tRNA, thereby allowing cells to survive low level serine CUG translation. Expression of this tRNA in S.cerevisiae induces the stress response which allows cells to acquire thermotolerance. We argue that acquisition of thermotolerance may represent a positive selection for this genetic code change by allowing yeasts to adapt to sudden changes in environmental conditions and therefore colonize new ecological niches.     

Optimization protein productivity of human interleukin-2 through codon usage,gene copy number and intracellular tRNA concentration in CHO cells

Transfer RNA (tRNA) abundance is one of the critical factors for the enhancement of protein productivity in prokaryotic and eukaryotic hosts. Gene copy number of tRNA and tRNA codon usage bias are generally used to match tRNA abundance of protein-expressing hosts and to optimize the codons of recombinant proteins. Because sufficient concentration of intracellular tRNA and optimized codons of recombinant proteins enhanced translation efficiency, we hypothesized that sufficient supplement of host’s tRNA improved protein productivity in mammalian cells. First, the small tRNA sequencing results of CHO-K1 cells showed moderate positive correlation with gene copy number and codon usage bias. Modification of human interleukin-2 (IL-2) through codons with high gene copy number and high codon usage bias (IL-2 HH, modified on Leu, Thr, Glu) significantly increased protein productivity in CHO-K1 cells. In contrast, modification through codons with relatively high gene copy number and low codon usage bias (IL-2 HL, modified on Ala, Thr, Val), or relatively low gene copy number and low codon usage bias (IL-2 LH, modified on Ala, Thr, Val) did not increase IL-2 productivity significantly. Furthermore, supplement of the alanine tRNA or threonine tRNA increased IL-2 productivity of IL-2 HL. In summary, we revealed a potential strategy to enhance productivity of recombinant proteins, which may be applied in production of protein drug or design of DNA vaccine.     

Selection on the codon bias ofChlamydomonas reinhardtii chloroplast genes and the plantpsbA gene

Plant chloroplast genes have a codon use that reflects the genome compositional bias of a high A+T content with the single exception of the highly translatedpsbA gene which codes for the photosystem II D1 protein. The codon usage of plantpsbA corresponds more closely to the limited tRNA population of the chloroplast and is very similar to the codon use observed in the chloroplast genes of the green algaChlamydomonas reinhardtii. This pattern of codon use may be an adaptation for increased translation efficiency. A correspondence between codon use of plantpsbA andChlamydomonas chloroplast genes and the tRNAs coded by the chloroplast genome, however, is not observed in all synonymous codon groups. It is shown here that the degree of correspondence between codon use and tRNA population in different synonymous groups is correlated with the second codon position composition. Synonymous groups with an A or T at the second codon position have a high representation of codons for which a complementary tRNA is coded by the chloroplast genome. Those with a G or C at the second position have an increased representation of codons that bind a chloroplast tRNA by wobble. It is proposed that the difference between synonymous groups in terms of codon adaptation to the tRNA population in plantpsbA andChlamydomonas chloroplast genes may be the result of differences in second position composition.     

Transformation systems of non-Saccharomyces yeasts

This review describes the transformation systems including vectors, replicons, genetic markers, transformation methods, vector stability, and copy numbers of 13 genera and 31 species of non-Saccharomyces yeasts. Schizosaccharomyces pombe was the first non-Saccharomyces yeast studied for transformation and genetics. The replicons of non-Saccharomyces yeast vectors are from native plasmids, chromosomal DNA, and mitochondrial DNA of Saccharomyces cerevisiae, non-Saccharomyces yeasts, protozoan, plant, and animal. Vectors such as YAC, YCp, YEp, YIp, and YRp were developed for non-Saccharomyces yeasts. Forty-two types of genes from bacteria, yeasts, fungi, and plant were used as genetic markers that could be classified into biosynthetic, dominant, and colored groups to construct non-Saccharomyces yeasts vectors. The LEU2 gene and G418 resistance gene are the two most popular markers used in the yeast transformation. All known transformation methods such as spheroplast-mediating method, alkaline ion treatment method, electroporation, trans-kingdom conjugation, and biolistics have been developed successfully for non-Saccharomyces yeasts, among which the first three are most widely used. The highest copy number detected from non-Saccharomyces yeasts is 60 copies in Kluyveromyces lactis. No general rule is known to illustrate the transformation efficiency, vector stability, and copy number, although factors such as vector composition, host strain, transformation method, and selective pressure might influence them.     

Codon optimizer: a freeware tool for codon optimization

Selection plays a major role in the determination of codon usage in all organisms studied so far. In highly expressed genes, a narrow set of codons is used and these codons correspond to the more abundant tRNA species. This minimizes the risk of tRNA depletion during translation. In fact, the codons in a gene may be true bottlenecks, especially in cases where foreign genes are expressed in a host in which the usage of codons in highly expressed genes does not resemble the usage of codons in the species from which the foreign gene originates. In such cases, it has been shown that substitution of rare codons in the introduced gene may increase the yield dramatically. In addition, replacement of rare codons might decrease the chance of misincorporation and protect the protein from premature turnover. Here, a piece of software is announced that calculates a codon-optimized sequence of any gene based on knowledge of highly expressed genes of a host. In addition, it calculates the codon adaptation index of the gene and identifies internal type II restriction sites of the optimized sequence. The program runs under Windows and is available as freeware for use in academia.     

Influence of the codon following the AUG initiation codon on the expression of a modified lacZ gene in Escherichia coli.

In a lacZ expression vector (pMC1403Plac), all 64 codons were introduced immediately 3' from the AUG initiation codon. The expression of the second codon variants was measured by immunoprecipitation of the plasmid-coded fusion proteins. A 15-fold difference in expression was found among the codon variants. No distinct correlation could be made with the level of tRNA corresponding to the codons and large differences were observed between synonymous codons that use the same tRNA. Therefore the effect of the second codon is likely to be due to the influence of its composing nucleotides, presumably on the structure of the ribosomal binding site. An analysis of the known sequences of a large number of Escherichia coli genes shows that the use of codons in the second position deviates strongly from the overall codon usage in E. coli. It is proposed that codon selection at the second position is not based on requirements of the gene product (a protein) but is determined by factors governing gene regulation at the initiation step of translation.     

Nature of selective constraints on synonymous codon usage of rice differs in GC-poor and GC-rich genes

Synonymous codon usage and cellular tRNA abundance are thought to be co-evolved in optimizing translational efficiencies in highly expressed genes. Here in this communication by taking the advantage of publicly available gene expression data of rice and Arabidopsis we demonstrated that tRNA gene copy number is not the only driving force favoring translational selection in all highly expressed genes of rice. We found that forces favoring translational selection differ between GC-rich and GC-poor classes of genes. Supporting our results we also showed that, in highly expressed genes of GC-poor class there is a perfect correspondence between majority of preferred codons and tRNA gene copy number that confers translational efficiencies to this group of genes. However, tRNA gene copy number is not fully consistent with models of translational selection in GC-rich group of genes, where constraints on mRNA secondary structure play a role to optimize codon usage in highly expressed genes.     

Probing fungal mitochondrial evolution with tRNA

Sequence data are now available for almost the entire complement of mitochondrial rRNAs from five fungi: Schizosaccharomyces pombe, Saccharomyces cerevisiae, Toropulis glabrata, Aspergillus nidulans and Neurospora crassa. Analysis of these data show that the five mitochondria can be related to a common ancestor. The unusually high similarity between some S. pombe mt tRNAs may be due to a process similar to gene conversion. Using the number of differences between tRNA pairs as a measure of the evolutionary rate the yeast-S. pombe branch has paradoxically a high nuclear rate and a low mt rate of evolution as compared with other branches in the phylogenetic tree. Finally the position of mt tRNA genes in S. pombe is abnormally distinct from gene orders in other mitochondria. All of the above factors must be taken into account when describing the relationship between these mitochondria.     

Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression.

The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.