Bayesian analysis suggests that most amino acid replacements in Drosophila are driven by positive selection

One of the principal goals of population genetics is to understand the processes by which genetic variation within species (polymorphism) becomes converted into genetic differences between species (divergence). In this transformation, selective neutrality, near neutrality, and positive selection may each play a role, differing from one gene to the next. Synonymous nucleotide sites are often used as a uniform standard of comparison across genes on the grounds that synonymous sites are subject to relatively weak selective constraints and so may, to a first approximation, be regarded as neutral. Synonymous sites are also interdigitated with nonsynonymous sites and so are affected equally by genomic context and demographic factors. Hence a comparison of levels of polymorphism and divergence between synonymous sites and amino acid replacement sites in a gene is potentially informative about the magnitude of selective forces associated with amino acid replacements. We have analyzed 56 genes in which polymorphism data from D. simulans are compared with divergence from a reference strain of D. melanogaster. The framework of the analysis is Bayesian and assumes that the distribution of selective effects (Malthusian fitnesses) is Gaussian with a mean that differs for each gene. In such a model, the average scaled selection intensity (gamma = N(e)s) of amino acid replacements eligible to become polymorphic or fixed is -7.31, and the standard deviation of selective effects within each locus is 6.79 (assuming homoscedasticity across loci). For newly arising mutations of this type that occur in autosomal or X-linked genes, the average proportion of beneficial mutations is 19.7%. Among the amino acid polymorphisms in the sample, the expected average proportion of beneficial mutations is 47.7%, and among amino acid replacements that become fixed the average proportion of beneficial mutations is 94.3%. The average scaled selection intensity of fixed mutations is +5.1. The presence of positive selection is pervasive with the single exception of kl-5, a Y-linked fertility gene. We find no evidence that a significant fraction of fixed amino acid replacements is neutral or nearly neutral or that positive selection drives amino acid replacements at only a subset of the loci. These results are model dependent and we discuss possible modifications of the model that might allow more neutral and nearly neutral amino acid replacements to be fixed.     

Aminoacylation of transfer RNAs with one and two amino acids

The detailed synthesis of (bis)aminoacyl-pdCpAs and the corresponding singly and tandemly activated tRNAs is reported. The synthetic pathway leading to these compounds has been validated for simple amino acid residues as well as for amino acids bearing more complex side chains. Protection/deprotection strategies are described. For the bisaminoacylated tRNAs, both the synthesis of tRNAs bearing the same amino acid residue at the 2' and 3' positions and tRNAs bearing two different aminoacyl moieties are reported. Further, it is shown that the tandemly activated tRNAs are able to participate in protein synthesis.     

Molecular divergence and phylogeny: rates and patterns of cytochrome b evolution in cranes

Analyses of complete cytochrome b sequences from all species of cranes (Aves: Gruidae) reveal aspects of sequence evolution in the early stages of divergence. These DNA sequences are > or = 89% identical, but expected departures from random substitution are evident. Silent, third- position pyrimidine transitions are the dominant substitution type, with transversion comprising only a small fraction of sequence differences. Substitution patterns are not clearly manifested until divergence has reached a moderate level (> 3%), as expected for a stochastic process. Variation in the frequency of mismatch types among lineages decreases at larger divergences, but the level of bias does not decay. Divergence varies up to fivefold among gene regions but is not correlated with structural domain. All protein structural domains except extramembrane 4 display < 20% variable residues. Regions corresponding to putative functional domains show the excepted conservation of amino acids, although the C-terminal portion of the Q0 reaction center displays several nonconservative replacements. Phylogenetic analyses incorporating substitution asymmetries produced mixed results. Distances estimated with multiple parameters (transition, codon-position, composition, and pyrimidine-transition biases) yielded identical additive tree topologies with comparable bootstrap values, all consistent with uncontroversial species relationships. Maximum likelihood analysis incorporating these biases, as well as equally weighted parsimony analysis, produced similar results. Static, differential weighting for parsimony did not improve the phylogenetic signal but produced unusual trees with low bootstraps. The overall rate of nucleotide substitution varies slightly but significantly among cranes, and calibration of distances against fossil dates suggests divergence rates of 0.7%-1.7% per million years.      

Comparative Analysis of Mitochondrial Genomes in <Emphasis Type="Italic">Bombina</Emphasis> (Anura; Bombinatoridae)

The complete mitochondrial genomes of two basal anurans, Bombina bombina and B. variegata (Anura; Bombinatoridae), were sequenced. The gene order of their mitochondrial DNA (mtDNA) is identical to that of canonical vertebrate mtDNA. In contrast, we show that there are structural differences in regulatory regions and protein coding genes between the mtDNA of these two closely related species. Corrected sequence divergence between the mtDNA of B. bombina and B. variegata amounts to 8.7% (2.3% divergence in amino acids). Comparisons with two East Asian congeners show that the control region contains two repeat regions, LV1 and LV2, present in all species except for B. bombina, in which LV2 has been secondarily lost. The rRNAs and tRNAs are characterized by low nucleotide divergence. The protein coding genes are considerably more disparate, although functional constraint is high but variable among genes, as evidenced by dN/dS ratios. A mtDNA phylogeny established the distribution of autapomorphic nonsynonomous substitutions in the mitogenomes of B. bombina and B. variegata. Nine of 98 nonsynonomous substitutions led to radical amino acid replacements that may alter mitochondrial protein function. Most radical substitutions were found in ND2, ND4, or ND5, encoding mitochondrial subunits of complex I of the electron transport system. The extensive divergence between the mitogenomes of B. bombina and B. variegata is discussed in terms of its possible role in impeding gene flow in natural hybrid zones between these two species.     

tRNAs: Cellular barcodes for amino acids

The role of tRNA in translating the genetic code has received considerable attention over the last 50 years, and we now know in great detail how particular amino acids are specifically selected and brought to the ribosome in response to the corresponding mRNA codon. Over the same period, it has also become increasingly clear that the ribosome is not the only destination to which tRNAs deliver amino acids, with processes ranging from lipid modification to antibiotic biosynthesis all using aminoacyl-tRNAs as substrates. Here we review examples of alternative functions for tRNA beyond translation, which together suggest that the role of tRNA is to deliver amino acids for a variety of processes that includes, but is not limited to, protein synthesis.     

Eukaryotic tRNA sequences present conserved and amino acid-specific structural signatures

Metazoan organisms have many tRNA genes responsible for decoding amino acids. The set of all tRNA genes can be grouped in sets of common amino acids and isoacceptor tRNAs that are aminoacylated by corresponding aminoacyl-tRNA synthetases. Analysis of tRNA alignments shows that, despite the high number of tRNA genes, specific tRNA sequence motifs are highly conserved across multicellular eukaryotes. The conservation often extends throughout the isoacceptors and isodecoders with, in some cases, two sets of conserved isodecoders. This study is focused on non-Watson–Crick base pairs in the helical stems, especially GoU pairs. Each of the four helical stems may contain one or more conserved GoU pairs. Some are amino acid specific and could represent identity elements for the cognate aminoacyl tRNA synthetases. Other GoU pairs are found in more than a single amino acid and could be critical for native folding of the tRNAs. Interestingly, some GoU pairs are anticodon-specific, and others are found in phylogenetically-specific clades. Although the distribution of conservation likely reflects a balance between accommodating isotype-specific functions as well as those shared by all tRNAs essential for ribosomal translation, such conservations may indicate the existence of specialized tRNAs for specific translation targets, cellular conditions, or alternative functions.     

Relationships Among Isoacceptor tRNAs Seems to Support the Coevolution Theory of the Origin of the Genetic Code

A new method for looking at relationships between nucleotide sequences has been used to analyze divergence both within and between the families of isoaccepting tRNA sets. A dendrogram of the relationships between 21 tRNA sets with different amino acid specificities is presented as the result of the analysis. Methionine initiator tRNAs are included as a separate set. The dendrogram has been interpreted with respect to the final stage of the evolutionary pathway with the development of highly specific tRNAs from ambiguous molecular adaptors. The location of the sets on the dendrogram was therefore analyzed in relation to hypotheses on the origin of the genetic code: the coevolution theory, the physicochemical hypothesis, and the hypothesis of ambiguity reduction of the genetic code. Pairs of 16 sets of isoacceptor tRNAs, whose amino acids are in biosynthetic relationships, occupied contiguous positions on the dendrogram, thus supporting the coevolution theory of the genetic code. Received: 4 May 1998 / Accepted: 11 July 1998     

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.     

Transfer RNA genes in the mitochondrial genome from a liverwort, Marchantia polymorpha: the absence of chloroplast-like tRNAs.

Twenty-nine genes for 27 species of tRNAs were deduced from the complete nucleotide sequence of the mitochondrial genome from a liverwort, Marchantia polymorpha. One to three species of tRNA genes corresponded to each of 20 amino acids including three species for leucine and arginine, two species for serine and glycine, and one for the rest of the amino acids. Interestingly, all tRNA genes were located in the semicircle of the liverwort mitochondrial genome except for the trnY and trnR genes. The region containing these tRNA genes was originally duplicated, and two trnR genes have diverged from each other. On the other hand, trnY and trnfM are present as two identical copies. The G:U and U:N wobbling between the first nucleotide of the anticodon and the third nucleotide of the codon permit the 27 tRNA identified species to translate almost all codons. However, at least two additional tRNA genes, trnl-GAU for AUY codon and trnT-UGU for ACR codon, are required to read all codons used in the liverwort mitochondrial genome. All of the identified tRNA genes are 'native' in liverwort mitochondria, not 'chloroplast-like' tRNAs as are found in the mitochondria of higher plants. This result implies that the tRNA gene transfer from chloroplast to mitochondrial genome in higher plants has occurred after the divergence from bryophytes.     

New insights into the incorporation of natural suppressor tRNAs at stop codons in Saccharomyces cerevisiae

Stop codon readthrough may be promoted by the nucleotide environment or drugs. In such cases, ribosomes incorporate a natural suppressor tRNA at the stop codon, leading to the continuation of translation in the same reading frame until the next stop codon and resulting in the expression of a protein with a new potential function. However, the identity of the natural suppressor tRNAs involved in stop codon readthrough remains unclear, precluding identification of the amino acids incorporated at the stop position. We established an in vivo reporter system for identifying the amino acids incorporated at the stop codon, by mass spectrometry in the yeast Saccharomyces cerevisiae. We found that glutamine, tyrosine and lysine were inserted at UAA and UAG codons, whereas tryptophan, cysteine and arginine were inserted at UGA codon. The 5′ nucleotide context of the stop codon had no impact on the identity or proportion of amino acids incorporated by readthrough. We also found that two different glutamine tRNA^Gln were used to insert glutamine at UAA and UAG codons. This work constitutes the first systematic analysis of the amino acids incorporated at stop codons, providing important new insights into the decoding rules used by the ribosome to read the genetic code.     

Presence of two transcribed malate synthase genes in an n-alkane-utilizing yeast, Candida tropicalis.

The presence of two genomic DNA regions encoding malate synthase (MS) was shown by Southern blot analysis of the genomic DNA from an n-alkane-assimilating yeast, Candida tropicalis, using a partial MS cDNA probe, in accordance with the fact that two types of partial MS cDNAs have previously been isolated. This was also confirmed by the restriction mapping of the two genes screened from the yeast lambda EMBL library. Nucleotide sequence analysis of the respective genomic DNAs, named MS-1 gene and MS-2 gene, revealed that both regions encoding MS had the same length of 1,653 base pairs, corresponding to 551 amino acids (molecular mass of MS-1, 62,448 Da; MS-2, 62,421 Da). Although 29 nucleotide pairs differed in the sequences of the coding regions, the number of amino acid replacements was only one: 159Asn (MS-1)----159Ser (MS-2). In the 5'-flanking regions, there were replacements of four nucleotide pairs, deletion of one pair, and insertion of four pairs. In spite of the fact that two genomic genes were present and transcribed, RNA blot analysis demonstrated that only one band (about 2 kb) was observable even when the carbon sources in the cultivation medium were changed. A comparison of the amino acid sequences was made with MSs of rape (Brassica napus L.), cucumber seed, pumpkin seed, Escherichia coli, and Hansenula polymorpha. A high homology was observed among these enzymes, the results indicating that the protein structure was relatively well conserved through the evolution of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fitness conferred by replaced amino acids declines with time

The fitness landscape of a locus, the array of fitnesses conferred by its alleles, can be affected by allele replacements at other loci, in the presence of epistatic interactions between loci. In a pair of diverging homologous proteins, the initially high probability that an amino acid replacement in one of them will make it more similar to the other declines with time, implying that the fitness landscapes of homologous sites diverge. Here, we use data on within-population non-synonymous polymorphisms and on amino acid replacements between species to study the dynamics, after an amino acid replacement, of the fitness of the ancestral amino acid, and show that selection against its restoration increases with time. This effect can be owing to increase of fitness conferred by the new amino acid occupying the site, and/or to decline of fitness conferred by the replaced amino acid. We show that the fitness conferred by the replaced amino acid rapidly declines, reaching a new lower steady-state level after approximately 20 per cent of amino acids in the protein get replaced. Therefore, amino acid replacements in evolving proteins are routinely involved in negative epistatic interactions with currently absent amino acids, and chisel off the unused parts of the fitness landscape.     

The Role of Initiator tRNAi met in Fidelity of Initiation of Protein Synthesis

The proper arrangement of amino acids in a protein determines its proper function, which is vital for the cellular metabolism. This indicates that the process of peptide bond formation requires high fidelity. One of the most important processes for this fidelity is kinetic proofreading. As biochemical experiments suggest that kinetic proofreading plays a major role in ensuring the fidelity of protein synthesis, it is not certain whether or not a misacylated tRNA would be corrected by kinetic proofreading during the peptide bond formation. Using 2-layered ONIOM (QM/MM) computational calculations, we studied the behavior of misacylated tRNAs and compared the results with these for cognate aminoacyl-tRNAs during the process of peptide bond formation to investigate the effect of nonnative amino acids on tRNAs. The difference between the behavior of initiator tRNA_i ^met compared to the one for the elongator tRNAs indicates that only the initiator tRNA_i ^met specifies the amino acid side chain.     

A comparison of mitochondrial tRNAs in five vertebrates

Sequences of several vertebrate mitochondrial tRNAs were aligned and compared. The comparisons were made in pairs of the tRNAs for an identical amino acid. There are 22 genes for different tRNAs in each vertebrate mitochondrial DNA. The closest similarities were between rat and mouse, the next were between mammals, and the widest difference was between human or rat and Xenopus laevis. However, there were very wide variations between different amino acids in each set of comparisons. The time lapse for each percent of difference greatly increased with evolutionary separation. Most of the nucleotide substitutions appeared to be neutral in character.Based on a presentation made at a workshop- Aminoacyl-tRNA Synthetases and the Evolution of the Genetic Code-held at Berkeley, CA, July 17–20, 1994     

Expansion of the Genetic Code Through the Use of Modified Bacterial Ribosomes

Biological protein synthesis is mediated by the ribosome, and employs ~20 proteinogenic amino acids as building blocks. Through the use of misacylated tRNAs, presently accessible by any of several strategies, it is now possible to employ in vitro and in vivo protein biosynthesis to elaborate proteins containing a much larger variety of amino acid building blocks. However, the incorporation of this broader variety of amino acids is limited to those species utilized by the ribosome. As a consequence, virtually all of the substrates utilized over time have been L-α-amino acids. In recent years, a variety of structural and biochemical studies have provided important insights into those regions of the 23S ribosomal RNA that are involved in peptide bond formation. Subsequent experiments, involving the randomization of key regions of 23S rRNA required for peptide bond formation, have afforded libraries of E. coli harboring plasmids with the rrnB gene modified in the key regions. Selections based on the use of modified puromycin derivatives with altered amino acids then identified clones uniquely sensitive to individual puromycin derivatives. These clones often recognized misacylated tRNAs containing altered amino acids similar to those in the modified puromycins, and incorporated the amino acid analogues into proteins. In this fashion, it has been possible to realize the synthesis of proteins containing D-amino acids, β-amino acids, phosphorylated amino acids, as well as long chain and cyclic amino acids in which the nucleophilic amino group is not in the α-position. Of special interest have been dipeptides and dipeptidomimetics of diverse utility.     

Role of tRNA amino acid-accepting end in aminoacylation and its quality control

Aminoacyl–tRNA synthetases (aaRSs) are remarkable enzymes that are in charge of the accurate recognition and ligation of amino acids and tRNA molecules. The greatest difficulty in accurate aminoacylation appears to be in discriminating between highly similar amino acids. To reduce mischarging of tRNAs by non-cognate amino acids, aaRSs have evolved an editing activity in a second active site to cleave the incorrect aminoacyl–tRNAs. Editing occurs after translocation of the aminoacyl–CCA₇₆ end to the editing site, switching between a hairpin and a helical conformation for aminoacylation and editing. Here, we studied the consequence of nucleotide changes in the CCA₇₆ accepting end of tRNA^Leu during the aminoacylation and editing reactions. The analysis showed that the terminal A₇₆ is essential for both reactions, suggesting that critical interactions occur in the two catalytic sites. Substitutions of C₇₄ and C₇₅ selectively decreased aminoacylation keeping nearly unaffected editing. These mutations might favor the regular helical conformation required to reach the editing site. Mutating the editing domain residues that contribute to CCA₇₆ binding reduced the aminoacylation fidelity leading to cell-toxicity in the presence of non-cognate amino acids. Collectively, the data show how protein synthesis quality is controlled by the CCA₇₆ homogeneity of tRNAs.     

Codon equilibrium I: Testing for homogeneous equilibrium

Summary We present theoretical considerations that suggest that synonymous-codon usage might be expected to be close to an equilibrium distribution given a very homogeneous process of silent substitution. By homogeneous we mean that substitution depends only on the two bases involved, so that 12 base-substitution rates completely describe the silent substitution process. We have developed a method of statistically testing for such homogeneous equilibrium and applied it to reported data on the codon usages of different classes of organisms. Weakly expressed bacterial sequences and both mammalian and nonmammalian eukaryotic sequences deviate significantly from a random pattern of codon usage, in the direction of homogeneous equilibrium. On the other hand, highly expressed bacterial sequences do not exhibit homogeneous equilibrium, which may be correlated with recent experimental results showing that they are optimized to accept the most abundant tRNAs. To examine the effect of amino acid replacements on the homogeneous model of silent substitution, we divided the amino acids with degenerate codes into two classes, those with high mutabilities and those with low, and performed the same analysis on bacterial and eukaryotic data sets. The codon sets of the highly mutable class of amino acids are not further from homogeneous equilibrium than are the codon sets of the class with low mutabilities. We also found for the eukaryotic data that these independent classes of codon sets show very similar equilibrium patterns. The various results suggest a high level of uniformity in the process of silent fixation in the different synonymous-codon sets, especially in eukaryotes.     

Codon equilibrium I: Testing for homogeneous equilibrium

We present theoretical considerations that suggest that synonymous-codon usage might be expected to be close to an equilibrium distribution given a very homogeneous process of silent substitution. By homogeneous we mean that substitution depends only on the two bases involved, so that 12 base-substitution rates completely describe the silent substitution process. We have developed a method of statistically testing for such homogeneous equilibrium and applied it to reported data on the codon usages of different classes of organisms. Weakly expressed bacterial sequences and both mammalian and nonmammalian eukaryotic sequences deviate significantly from a random pattern of codon usage, in the direction of homogeneous equilibrium. On the other hand, highly expressed bacterial sequences do not exhibit homogeneous equilibrium, which may be correlated with recent experimental results showing that they are optimized to accept the most abundant tRNAs. To examine the effect of amino acid replacements on the homogeneous model of silent substitution, we divided the amino acids with degenerate codes into two classes, those with high mutabilities and those with low, and performed the same analysis on bacterial and eukaryotic data sets. The codon sets of the highly mutable class of amino acids are not further from homogeneous equilibrium than are the codon sets of the class with low mutabilities. We also found for the eukaryotic data that these independent classes of codon sets show very similar equilibrium patterns. The various results suggest a high level of uniformity in the process of silent fixation in the different synonymous-codon sets, especially in eukaryotes.