Selection of aminoacyl-tRNAs at sense codons: the size of the tRNA variable loop determines whether the immediate 3' nucleotide to the codon has a context effect.

Codon context can affect translational efficiency by several molecular mechanisms. The base stacking interactions between a codon-anticodon complex and the neighboring nucleotide immediately 3' can facilitate translation by amber suppressors and the tRNA structure is also known to modulate the sensitivity to context. In this study the relative rates of aminoacyl-tRNA selection were measured at four sense codons (UGG, CUC, UUC and UCA), in all four 3' nucleotide contexts, through direct competition with a programmed frameshift at a site derived from the release factor 2 gene. Two codons (UGG and UUC) are read by tRNAs with small variable regions and their rates of aminoacyl-tRNA selection correlated with the potential base stacking strength of the 3' neighboring nucleotide. The other two codons (CUC and UCA) are read by tRNAs with large variable regions and the rate of selection of the aminoacyl-tRNAs in these cases varied little among the four contexts. Re-examination of published data on amber suppression also revealed an inverse correlation between context sensitivity and the size of the variable region. Collectively the data suggest that a large variable loop in a tRNA decreases the influence of the 3' context on tRNA selection, probably by strengthening tRNA-ribosomal interactions.     

Expanding the genetic code: selection of efficient suppressors of four-base codons and identification of "shifty" four-base codons with a library approach in Escherichia coli

Naturally occurring tRNA mutants are known that suppress +1 frameshift mutations by means of an extended anticodon loop, and a few have been used in protein mutagenesis. In an effort to expand the number of possible ways to uniquely and efficiently encode unnatural amino acids, we have devised a general strategy to select tRNAs with the ability to suppress four-base codons from a library of tRNAs with randomized 8 or 9 nt anticodon loops. Our selectants included both known and novel suppressible four-base codons and resulted in a set of very efficient, non-cross-reactive tRNA/four-base codon pairs for AGGA, UAGA, CCCU and CUAG. The most efficient four-base codon suppressors had Watson-Crick complementary anticodons, and the sequences of the anticodon loops outside of the anticodons varied with the anticodon. Additionally, four-base codon reporter libraries were used to identify "shifty" sites at which +1 frameshifting is most favorable in the absence of suppressor tRNAs in Escherichia coli. We intend to use these tRNAs to explore the limits of unnatural polypeptide biosynthesis, both in vitro and eventually in vivo. In addition, this selection strategy is being extended to identify novel five- and six-base codon suppressors.     

Five-base codons for incorporation of nonnatural amino acids into proteins

Extension of the genetic code for the introduction of nonnatural amino acids into proteins was examined by using five-base codon–anticodon pairs. A streptavidin mRNA containing a CGGUA codon at the Tyr54 position and a tRNA_UACCG chemically aminoacylated with a nonnatural amino acid were added to an Escherichia coli in vitro translation system. Western blot analysis indicated that the CGGUA codon is decoded by the aminoacyl-tRNA containing the UACCG anticodon. HPLC analysis of the tryptic fragment of the translation product revealed that the nonnatural amino acid was incorporated corresponding to the CGGUA codon without affecting the reading frame adjacent to the CGGUA codon. Another 15 five-base codons CGGN₁N₂, where N₁ and N₂ indicate one of four nucleotides, were also successfully decoded by aminoacyl-tRNAs containing the complementary five-base anticodons. These results provide a novel strategy for nonnatural mutagenesis as well as a novel insight into the mechanism of frameshift suppression.     

Discharging tRNAs: a tug of war between translation and detoxification in Escherichia coli

Translation is a central cellular process and is optimized for speed and fidelity. The speed of translation of a single codon depends on the concentration of aminoacyl-tRNAs. Here, we used microarray-based approaches to analyze the charging levels of tRNAs in Escherichia coli growing at different growth rates. Strikingly, we observed a non-uniform aminoacylation of tRNAs in complex media. In contrast, in minimal medium, the level of aminoacyl-tRNAs is more uniform and rises to approximately 60%. Particularly, the charging level of tRNA^Ser, tRNA^Cys, tRNA^Thr and tRNA^His is below 50% in complex medium and their aminoacylation levels mirror the degree that amino acids inhibit growth when individually added to minimal medium. Serine is among the most toxic amino acids for bacteria and tRNAs^Ser exhibit the lowest charging levels, below 10%, at high growth rate although intracellular serine concentration is plentiful. As a result some serine codons are among the most slowly translated codons. A large fraction of the serine is most likely degraded by L-serine-deaminase, which competes with the seryl-tRNA-synthetase that charges the tRNAs^Ser. These results indicate that the level of aminoacylation in complex media might be a competition between charging for translation and degradation of amino acids that inhibit growth.     

Effect of replacing uridine 33 in yeast tRNAPhe on the reaction with ribosomes

We have determined several kinetic parameters for the reaction of poly(U)-programmed ribosomes with ternary complexes of elongation factor Tu, GTP, and yeast Phe-tRNA analogs with different bases substituted for uridine in position 33. These analogs test whether disruption of the hydrogen bonds normally formed by uridine 33 and steric crowding in the anticodon loop are detrimental to tRNA function on the ribosome. Single-turnover kinetic studies of the reaction of these ternary complexes with ribosomes show that these Phe-tRNA analogs decrease the apparent rate of GTP hydrolysis (kGTP) and the ratio of peptide formed to GTP hydrolyzed. Thus, the substitution of uridine 33 affects not only the selection of a ternary complex by the ribosome but also the selection of an aminoacyl-tRNA in the proofreading reaction. The effects become greater as first one, and then the other, H-bond is disrupted. Steric crowding in the anticodon loop is also important, but does not have as great an effect on the rate constants. An analysis of the elementary rate constants which comprise the rate constant, kGTP, demonstrates that the reduction in kGTP results from a decreased rate of ternary complex association with the ribosome (k1) and that there is little or no effect on the rate of GTP cleavage (k2). An analysis of the rate constants involved in proofreading shows that all the modified (tRNAs have increased rates of aminoacyl-tRNA rejection (k4) but that the rate of peptide bond formation (k3) is unaffected.     

The presence of pseudouridine in the anticodon alters the genetic code: a possible mechanism for assignment of the AAA lysine codon as asparagine in echinoderm mitochondria

It has been inferred from DNA sequence analyses that in echinoderm mitochondria not only the usual asparagine codons AAU and AAC, but also the usual lysine codon AAA, are translated as asparagine by a single mitochondrial (mt) tRNAAsn with the anticodon GUU. Nucleotide sequencing of starfish mt tRNAAsn revealed that the anticodon is GPsiU, U35 at the anticodon second position being modified to pseudouridine (Psi). In contrast, mt tRNALys, corresponding to another lysine codon, AAG, has the anticodon CUU. mt tRNAs possessing anti-codons closely related to that of tRNAAsn, but responsible for decoding only two codons each-tRNAHis, tRNAAsp and tRNATyr-were found to possess unmodified U35 in all cases, suggesting the importance of Psi35 for decoding the three codons. Therefore, the decoding capabilities of two synthetic Escherichia coli tRNAAla variants with the anticodon GPsiU or GUU were examined using an E.coli in vitro translation system. Both tRNAs could translate not only AAC and AAU with similar efficiency, but also AAA with an efficiency that was approximately 2-fold higher in the case of tRNAAlaGPsiU than tRNAAlaGUU. These findings imply that Psi35 of echinoderm mt tRNAAsn actually serves to decode the unusual asparagine codon AAA, resulting in the alteration of the genetic code in echinoderm mitochondria.     

Preferential codon usage in prokaryotic genes: the optimal codon-anticodon interaction energy and the selective codon usage in efficiently expressed genes

By considering the nucleotide sequence of several highly expressed coding regions in bacteriophage MS2 and mRNAs from Escherichia coli, it is possible to deduce some rules which govern the selection of the most appropriate synonymous codons NNU or NNC read by tRNAs having GNN, QNN or INN as anticodon. The rules fit with the general hypothesis that an efficient in-phase translation is facilitated by proper choice of degenerate codewords promoting a codon-anticodon interaction with intermediate strength (optimal energy) over those with very strong or very weak interaction energy. Moreover, codons corresponding to minor tRNAs are clearly avoided in these efficiently expressed genes. These correlations are clearcut in the normal reading frame but not in the corresponding frameshift sequences +1 and +2. We hypothesize that both the optimization of codon-anticodon interaction energy and the adaptation of the population to codon frequency or vice versa in highly expressed mRNAs of E. coli are part of a strategy that optimizes the efficiency of translation. Conversely, codon usage in weakly expressed genes such as repressor genes follows exactly the opposite rules. It may be concluded that, in addition to the need for coding an amino acid sequence, the energetic consideration for codon-anticodon pairing, as well as the adaptation of codons to the tRNA population, may have been important evolutionary constraints on the selection of the optimal nucleotide sequence.     

Missense and nonsense suppressors can correct frameshift mutations

Missense and nonsense suppressor tRNAs, selected for their ability to read a new triplet codon, were observed to suppress one or more frameshift mutations in trpA of Escherichia coli. Two of the suppressible frameshift mutants, trpA8 and trpA46AspPR3, were cloned, sequenced, and found to be of the +1 type, resulting from the insertion of four nucleotides and one nucleotide, respectively. Twenty-two suppressor tRNAs were examined, 20 derived from one of the 3 glycine isoacceptor species, one from lysT, and one from trpT. The sequences of all but four of the mutant tRNAs are known, and two of those four were converted to suppressor tRNAs that were subsequently sequenced. Consideration of the coding specificities and anticodon sequences of the suppressor tRNAs does not suggest a unitary mechanism of frameshift suppression. Rather, the results indicate that different suppressors may shift frame according to different mechanisms. Examination of the suppression windows of the suppressible frameshift mutations indicates that some of the suppressors may work at cognate codons, either in the 0 frame or in the +1 frame, and others may act at noncognate codons (in either frame) by some as-yet-unspecified mechanism. Whatever the mechanisms, it is clear that some +1 frameshifting can occur at non-monotonous sequences. A striking example of a frameshifting missense suppressor is a mutant lysine tRNA that differs from wild-type lysine tRNA by only a single base in the amino acid acceptor stem, a C to U70 transition that results in a G.U base pair. It is suggested that when this mutant lysine tRNA reads its cognate codon, AAA, the presence of the G.U base pair sometimes leads either to a conformational change in the tRNA or to an altered interaction with some component of the translation machinery involved in translocation, resulting in a shift of reading frame. In general, the results indicate that translocation is not simply a function of anticodon loop size, that different frameshifting mechanisms may operate with different tRNAs, and that conformational features, some far removed from the anticodon region, are involved in maintaining fidelity in translocation.     

On the role of the starved codon and the takeoff site in ribosome bypassing in Escherichia coli

Translating ribosomes can skip over stretches of messenger RNA and resume protein chain elongation after a "bypassed" region. We have previously shown that limitation for isoleucyl-tRNA can initiate a ribosome bypass when an AUA codon is in the ribosomal A-site. We have now generalized this effect to other "hungry" codons calling for four different limiting aminoacyl-tRNA species, suggesting that a pause at any A-site will have this effect. We have assessed bypassing in a large family of reporters with nearly every different triplet in the "takeoff site", i.e. the P-site on the 5' side of the hungry codon, and an identical "landing site" codon 16 nucleotides downstream. The different takeoff sites vary over a factor of 50 in bypassing proficiency. At least part of this variation appears to reflect stability of the codon Colon, two colons anticodon interaction at the takeoff site, as indicated by the following: (a) the bypassing proficiency of different tRNAs shows a rough correlation with the frequency of A Colon, two colons U as opposed to G Colon, two colons C pairs in the codon Colon, two colons anticodon association; (b) specific tRNAs bypass more frequently from codons ending in U than from their synonym ending in C; (c) an arginine tRNA with Inosine in the wobble position which reads CGU, CGC, and CGA bypasses much more frequently from the last codon than the first two synonyms.     

Direct analysis of aminoacylation levels of tRNAs in vivo. Application to studying recognition of Escherichia coli initiator tRNA mutants by glutaminyl-tRNA synthetase.

We describe the use of a gel electrophoretic method for measuring the levels of aminoacylation in vivo of mutant Escherichia coli initiator tRNAs, which are substrates for E. coli glutaminyl-tRNA synthetase (GlnRS) due to an anticodon sequence change. Using this method, we have compared the effects of introducing further mutations in the acceptor stem, at base pairs 1:72, 2:71, and 3:70 and discriminator base 73, on the recognition of these tRNAs by E. coli GlnRS in vitro and in vivo. The effects of the acceptor stem mutations on the kinetic parameters for aminoacylation of the mutant tRNAs in vitro are consistent with interactions seen between this region of tRNA and GlnRS in the crystal structure of tRNA(Gln). GlnRS complex. Except for one mutant, the observed levels of aminoacylation of the mutant tRNAs in vivo agree with those expected on the basis of the kinetic parameters obtained in vitro. We have also measured the relative amounts of aminoacyl-tRNAs for the various mutants and their activities in suppression of an amber codon in vivo. We find that there is, in general, a good correlation between the relative amounts of aminoacyl-tRNAs and their activities in suppression.     

Genomics and the evolution of aminoacyl-tRNA synthesis.

Translation is the process by which ribosomes direct protein synthesis using the genetic information contained in messenger RNA (mRNA). Transfer RNAs (tRNAs) are charged with an amino acid and brought to the ribosome, where they are paired with the corresponding trinucleotide codon in mRNA. The amino acid is attached to the nascent polypeptide and the ribosome moves on to the next codon. Thus, the sequential pairing of codons in mRNA with tRNA anticodons determines the order of amino acids in a protein. It is therefore imperative for accurate translation that tRNAs are only coupled to amino acids corresponding to the RNA anticodon. This is mostly, but not exclusively, achieved by the direct attachment of the appropriate amino acid to the 3'-end of the corresponding tRNA by the aminoacyl-tRNA synthetases. To ensure the accurate translation of genetic information, the aminoacyl-tRNA synthetases must display an extremely high level of substrate specificity. Despite this highly conserved function, recent studies arising from the analysis of whole genomes have shown a significant degree of evolutionary diversity in aminoacyl-tRNA synthesis. For example, non-canonical routes have been identified for the synthesis of Asn-tRNA, Cys-tRNA, Gln-tRNA and Lys-tRNA. Characterization of non-canonical aminoacyl-tRNA synthesis has revealed an unexpected level of evolutionary divergence and has also provided new insights into the possible precursors of contemporary aminoacyl-tRNA synthetases.     

Novel anticodon composition of transfer RNAs in Micrococcus luteus, a bacterium with a high genomic G + C content. Correlation with codon usage.

The number and relative amount of isoacceptor tRNAs for each amino acid in Micrococcus luteus, a Gram-positive bacterium with high genomic G + C content, have been determined by sequencing their anticodon loop and its adjacent regions and by selective labelling of tRNAs. Thirty-one tRNA species with 29 different anticodon sequences have been detected. All the tRNAs have G or C at the anticodon first position except for tRNA(ICGArg) and tRNA(NGASer), in response to the abundant usage of NNC and NNG codons. No tRNA with the anticodon UNN capable of translating codon NNA has been detected, in accordance with a very low or zero usage of NNA codons. The relative amount of isoacceptor tRNAs for an amino acid determined by selective labelling strongly correlates with usage of the corresponding codons. On the basis of these and other observations in this and other eubacterial species, we conclude that the relative amount and anticodon composition of isoacceptor tRNA species are flexible, and their changes are mainly adaptive phenomena that have been primarily affected by codon usage, which in turn is affected by directional mutation pressure.     

Decoding with the A:I wobble pair is inefficient.

tRNAs with inosine (I) in the first position read three codons ending in U, C and A. However, A-ending codons read with I are rarely used. In Escherichia coli, CGA/U/C are all read solely by tRNAICGArg. CGU and CGC are very common codons, but CGA is very rare. Three independent in vivo assays show that translation of CGA is relatively inefficient. In the first, nine tandem CGA cause a strong rho-mediated polar effect on expression of a lacZ reporter gene. The inhibition is made more extreme by a mutation in ribosomal protein S12 (rpsL), which indicates that ribosomal binding by tRNAICGArg is slow and/or unstable in the CGA cluster. The second assay, in which codons are substituted for the regulatory UGA of the RF2 frameshift, confirms that aa-tRNA selection is slow and/or unstable at CGA. In the third assay, CGA is found to be a poor 5' context for amber suppression, which suggests that an A:I base pair in the P site can interfere with translation of a codon in the A site. Two possible errors, frameshifting and premature termination by RF2, are not significant causes for inefficiency at CGA. It is concluded that the A:I pair destabilizes codon:anticodon complexes during two successive ribosomal cycles, and it is suggested that these properties contribute to the rare usage of codons read with the A:I base pair.     

Conflict between translation initiation and elongation in vertebrate mitochondrial genomes

The strand-biased mutation spectrum in vertebrate mitochondrial genomes results in an AC-rich L-strand and a GT-rich H-strand. Because the L-strand is the sense strand of 12 protein-coding genes out of the 13, the third codon position is overall strongly AC-biased. The wobble site of the anticodon of the 22 mitochondrial tRNAs is either U or G to pair with the most abundant synonymous codon, with only one exception. The wobble site of Met-tRNA is C instead of U, forming the Watson-Crick match with AUG instead of AUA, the latter being much more frequent than the former. This has been attributed to a compromise between translation initiation and elongation; i.e., AUG is not only a methionine codon, but also an initiation codon, and an anticodon matching AUG will increase the initiation rate. However, such an anticodon would impose selection against the use of AUA codons because AUA needs to be wobble-translated. According to this translation conflict hypothesis, AUA should be used relatively less frequently compared to UUA in the UUR codon family. A comprehensive analysis of mitochondrial genomes from a variety of vertebrate species revealed a general deficiency of AUA codons relative to UUA codons. In contrast, urochordate mitochondrial genomes with two tRNA(Met) genes with CAU and UAU anticodons exhibit increased AUA codon usage. Furthermore, six bivalve mitochondrial genomes with both of their tRNA-Met genes with a CAU anticodon have reduced AUA usage relative to three other bivalve mitochondrial genomes with one of their two tRNA-Met genes having a CAU anticodon and the other having a UAU anticodon. We conclude that the translation conflict hypothesis is empirically supported, and our results highlight the fine details of selection in shaping molecular evolution.     

Effect of trypsin modification of the Escherichia coli elongation factor Tu on the ternary complex with aminoacyl-tRNA

The ribonuclease resistance assay has been used to probe the effect of trypsin modification of the Escherichia coli elongation factor Tu X GTP on the interaction with E. coli aminoacyl-tRNAs. First, the equilibrium dissociation constant of the trypsin-modified Tu X GTP X Thr-tRNA complex was determined to be 2.3 (0.1) X 10(-5)M at 4 degrees C, pH 7.4. Second, binding of 17 of 20 noninitiator aminoacyl-tRNAs and four sets of purified isoacceptor tRNAs to the modified protein was measured. At 4 degrees C, the complex stabilities vary 500-fold over the range of aminoacyl-tRNAs, with Gln-tRNA forming the strongest ternary complex and Val-tRNA, the weakest. The results are compared to a similar study of ternary complex formation using intact elongation factor Tu X GTP, and the major differences are discussed. An analysis of both data sets, particularly that for the leucine isoacceptor tRNAs, suggests that the trypsin modification of elongation factor Tu X GTP disrupts a region of protein that is involved with the aminoacyl side chain rather than that of the acceptor stem helix region of the aminoacyl-tRNA.     

Common location of determinants in initiator transfer RNAs for initiator-elongator discrimination in bacteria and in eukaryotes

Initiator tRNAs are used exclusively for initiation of protein synthesis and not for elongation. We show that both Escherichia coli and eukaryotic initiator tRNAs have negative determinants, at the same positions, that block their activity in elongation. The primary negative determinant in E. coli initiator tRNA is the C1xA72 mismatch at the end of the acceptor stem. The primary negative determinant in eukaryotic initiator tRNAs is located in the TPsiC stem, whereas a secondary negative determinant is the A1:U72 base pair at the end of the acceptor stem. Here we show that E. coli initiator tRNA also has a secondary negative determinant for elongation and that it is the U50.G64 wobble base pair, located at the same position in the TPsiC stem as the primary negative determinant in eukaryotic initiator tRNAs. Mutation of the U50.G64 wobble base pair to C50:G64 or U50:A64 base pairs increases the in vivo amber suppressor activity of initiator tRNA mutants that have changes in the acceptor stem and in the anticodon sequence necessary for amber suppressor activity. Binding assays of the mutant aminoacyl-tRNAs carrying the C50 and A64 changes to the elongation factor EF-Tu.GTP show marginally higher affinity of the C50 and A64 mutant tRNAs and increased stability of the EF-Tu.GTP. aminoacyl-tRNA ternary complexes. Other results show a large effect of the amino acid attached to a tRNA, glutamine versus methionine, on the binding affinity toward EF-Tu.GTP and on the stability of the EF-Tu.GTP.aminoacyl-tRNA ternary complex.     

Alignment/misalignment hypothesis for tRNA selection by the ribosome

Transfer RNAs (tRNAs) are the adaptor molecules that allow the ribosome to decode genetic information during protein synthesis. During decoding, the ribosome must chose the tRNA whose anticodon corresponds to the codon inscribed in the messenger RNA to incorporate the correct amino acid into the growing polypeptide chain. Fidelity is improved dramatically by a GTP hydrolysis event. Information about the correctness of the anticodon must be sent from the decoding center to the elongation factor, EF-Tu, where the GTP hydrolysis takes place. A second discrimination event entails the accommodation of the aminoacyl-tRNA into its fully bound A/A state inside the ribosome. Here, we present a hypothesis for a specific mechanism of signal transduction through the tRNA, which operates during GTPase activation and accommodation. We propose that the rigidity of the tRNA plays an important role in the transmission of the decoding signal. While the tRNA must flex during binding and accommodation, its anisotropic stiffness enables precise positioning of the acceptor arm in the A/T state, the A/A state and the accommodation corridor. Correct alignment will result in optimal GTPase activation and accommodation rates. Incorrect tRNAs, however, whose anticodons are misaligned, will also have acceptor arms that are misaligned, resulting in sub-optimal GTPase activation and accommodation rates. In the case of GTPase activation, it is possible that the misalignment of the acceptor arm affects the rate directly, by altering the conformational change of the switch region of EF-Tu, or indirectly, by changing the alignment of EF-Tu with respect to the sarcin-ricin loop (SRL) of the large ribosomal subunit.     

In vitro selection of tRNAs for efficient four-base decoding to incorporate non-natural amino acids into proteins in an Escherichia coli cell-free translation system

Position-specific incorporation of non-natural amino acids into proteins is a useful technique in protein engineering. In this study, we established a novel selection system to obtain tRNAs that show high decoding activity, from a tRNA library in a cell-free translation system to improve the efficiency of incorporation of non-natural amino acids into proteins. In this system, a puromycin-tRNA conjugate, in which the 3'-terminal A unit was replaced by puromycin, was used. The puromycin-tRNA conjugate was fused to a C-terminus of streptavidin through the puromycin moiety in the ribosome. The streptavidin-puromycin-tRNA fusion molecule was collected and brought to the next round after amplification of the tRNA sequence. We applied this system to select efficient frameshift suppressor tRNAs from a tRNA library with a randomly mutated anticodon loop derived from yeast tRNA CCCG Phe. After three rounds of the selection, we obtained novel frameshift suppressor tRNAs which had high decoding activity and good orthogonality against endogenous aminoacyl-tRNA synthetases. These results demonstrate that the in vitro selection system developed here is useful to obtain highly active tRNAs for the incorporation of non-natural amino acid from a tRNA library.     

Comparison of codon usage and tRNAs in mitochondrial genomes of Candida species

To gain insight into the nature of the mitochondrial genomes (mtDNA) of different Candida species, the synonymous codon usage bias of mitochondrial protein coding genes and the tRNAs in C. albicans, C. parapsilosis, C. stellata, C. glabrata and the closely related yeast Saccharomyces cerevisiae were analyzed. Common features of the mtDNA in Candida species are a strong A+T pressure on protein coding genes, and insufficient mitochondrial tRNA species are encoded to perform protein synthesis. The wobble site of the anticodon is always U for the NNR (NNA and NNG) codon families, which are dominated by A-ending codons, and always G for the NNY (NNC and NNU) codon families, which is dominated by U-ending codons, and always U for the NNN (NNA, NNU, NNC and NNG) codon families, which are dominated by A-ending codons and U-ending codons. Patterns of synonymous codon usage of Candida species can be classified into three groups: (1) optimal codon-anticodon usage, Glu, Lys, Leu (translated by anti-codon UAA), Gln, Arg (translated by anti-codon UCU) and Trp are containing NNR codons. NNA, whose corresponding tRNA is encoded in the mtDNA, is used preferentially. (2) Non-optimal codon-anticodon usage, Cys, Asp, Phe, His, Asn, Ser (translated by anti-codon GCU) and Tyr are containing NNY codons. The NNU codon, whose corresponding tRNA is not encoded in the mtDNA, is used preferentially. (3) Combined codon-anticodon usage, Ala, Gly, Leu (translated by anti-codon UAG), Pro, Ser (translated by anti-codon UGA), Thr and Val are containing NNN codons. NNA (tRNA encoded in the mtDNA) and NNU (tRNA not encoded in the mtDNA) are used preferentially. In conclusion, we propose that in Candida species, codons containing A or U at third position are used preferentially, regardless of whether corresponding tRNAs are encoded in the mtDNA. These results might be useful in understanding the common features of the mtDNA in Candida species and patterns of synonymous codon usage.