Do Amino Acid Biosynthetic Costs Constrain Protein Evolution in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>?

Prokaryotic organisms preferentially utilize less energetically costly amino acids in highly expressed genes. Studies have shown that the proteome of Saccharomyces cerevisiae also exhibits this behavior, but only in broad terms. This study examines the question of metabolic efficiency as a proteome-shaping force at a finer scale, examining whether trends consistent with cost minimization as an evolutionary force are present independent of protein function and amino acid physicochemical property, and consistently with respect to amino acid biosynthetic costs. Inverse correlations between the average amino acid biosynthetic cost of the protein product and the levels of gene expression in S. cerevisiae are consistent with natural selection to minimize costs. There are, however, patterns of amino acid usage that raise questions about the strength (and possibly the universality) of this selective force in shaping S. cerevisiae’s proteome.     

A comparison of the composition of silk proteins produced by spiders and insects.

Proteins that are highly expressed and composed of amino acids that are costly to synthesize are likely to place a greater drain on an organism's energy resources than proteins that are composed of ingested amino acids or ones that are metabolically simple to produce. Silks are highly expressed proteins produced by all spiders and many insects. We compared the metabolic costs of silks spun by arthropods by calculating the amount of ATP required to produce their component amino acids. Although a definitive conclusion requires detailed information on the dietary pools of amino acids available to arthropods, on the basis of the central metabolic pathways, silks spun by herbivorous, Lepidoptera larvae require significantly less ATP to synthesize than the dragline silks spun by predatory spiders. While not enough data are available to draw a statistically based conclusion, comparison of homologous silks across ancestral and derived taxa of the Araneoidea seems to suggest an evolutionary trend towards reduced silk costs. However, comparison of the synthetic costs of dragline silks across all araneomorph spiders suggests a complicated evolutionary pattern that cannot be attributed to phylogenetic position alone. We propose that the diverse silk-producing systems of the araneoid spiders (including three types of protein glues and three types of silk fibroin), evolved through intra-organ competition and that taxon-specific differences in the composition of silks drawn from homologous glands may reflect limited or fluctuating amino acid availability. The different functional properties of spider silks may be a secondary result of selection acting on different polypeptide templates.     

Amino acid cost and codon-usage biases in 6 prokaryotic genomes: a whole-genome analysis

For most prokaryotic organisms, amino acid biosynthesis represents a significant portion of their overall energy budget. The difference in the cost of synthesis between amino acids can be striking, differing by as much as 7-fold. Two prokaryotic organisms, Escherichia coli and Bacillus subtilis, have been shown to preferentially utilize less costly amino acids in highly expressed genes, indicating that parsimony in amino acid selection may confer a selective advantage for prokaryotes. This study confirms those findings and extends them to 4 additional prokaryotic organisms: Chlamydia trachomatis, Chlamydophila pneumoniae AR39, Synechocystis sp. PCC 6803, and Thermus thermophilus HB27. Adherence to codon-usage biases for each of these 6 organisms is inversely correlated with a coding region's average amino acid biosynthetic cost in a fashion that is independent of chemoheterotrophic, photoautotrophic, or thermophilic lifestyle. The obligate parasites C. trachomatis and C. pneumoniae AR39 are incapable of synthesizing many of the 20 common amino acids. Removing auxotrophic amino acids from consideration in these organisms does not alter the overall trend of preferential use of energetically inexpensive amino acids in highly expressed genes.     

Metabolic and Translational Efficiency in Microbial Organisms

Metabolic efficiency, as a selective force shaping proteomes, has been shown to exist in Escherichia coli and Bacillus subtilis and in a small number of organisms with photoautotrophic and thermophilic lifestyles. Earlier attempts at larger-scale analyses have utilized proxies (such as molecular weight) for biosynthetic cost, and did not consider lifestyle or auxotrophy. This study extends the analysis to all currently sequenced microbial organisms that are amenable to these analyses while utilizing lifestyle specific amino acid biosynthesis pathways (where possible) to determine protein production costs and compensating for auxotrophy. The tendency for highly expressed proteins (with adherence to codon usage bias as a proxy for expressivity) to utilize less biosynthetically expensive amino acids is taken as evidence of cost selection. A comprehensive analysis of sequenced genomes to identify those that exhibit strong translational efficiency bias (389 out of 1,700 sequenced organisms) is also presented.     

The significance of nitrogen cost minimization in proteomes of marine microorganisms

Marine microorganisms thrive under low levels of nitrogen (N). N cost minimization is a major selective pressure imprinted on open-ocean microorganism genomes. Here we show that amino-acid sequences from the open ocean are reduced in N, but increased in average mass compared with coastal-ocean microorganisms. Nutrient limitation exerts significant pressure on organisms supporting the trade-off between N cost minimization and increased average mass of amino acids that is a function of increased A+T codon usage. N cost minimization, especially of highly expressed proteins, reduces the total cellular N budget by 2.7–10% this minimization in combination with reduction in genome size and cell size is an evolutionary adaptation to nutrient limitation. The biogeochemical and evolutionary precedent for these findings suggests that N limitation is a stronger selective force in the ocean than biosynthetic costs and is an important evolutionary strategy in resource-limited ecosystems.     

Reinvestigating the codon and amino acid usage of S. cerevisiae genome: a new insight from protein secondary structure analysis

Biased usage of synonymous codons has been elucidated under the perspective of cellular tRNA abundance for quite a long time now. Taking advantage of publicly available gene expression data for Saccharomyces cerevisiae, a systematic analysis of the codon and amino acid usages in two different coding regions corresponding to the regular (helix and strand) as well as the irregular (coil) protein secondary structures, have been performed. Our analyses suggest that apart from tRNA abundance, mRNA folding stability is another major evolutionary force in shaping the codon and amino acid usage differences between the highly and lowly expressed genes in S. cerevisiae genome and surprisingly it depends on the coding regions corresponding to the secondary structures of the encoded proteins. This is obviously a new paradigm in understanding the codon usage in S. cerevisiae. Differential amino acid usage between highly and lowly expressed genes in the regions coding for the irregular protein secondary structure in S. cerevisiae is expounded by the stability of the mRNA folded structure. Irrespective of the protein secondary structural type, the highly expressed genes always tend to encode cheaper amino acids in order to reduce the overall biosynthetic cost of production of the corresponding protein. This study supports the hypothesis that the tRNA abundance is a consequence of and not a reason for the biased usage of amino acid between highly and lowly expressed genes.     

Genes in S. cerevisiae encoding proteins with domains homologous to the mammalian ras proteins

The ras genes, which were first identified by their presence in RNA tumor viruses and which belong to a highly conserved gene family in vertebrates, have two close homologs in yeast, detectable by Southern blotting. We have cloned both genes (RAS1 and RAS2) from plasmid libraries and determined the complete nucleotide sequence of their coding regions. They encode proteins with nearly 90% homology to the first 80 positions of the mammalian ras proteins, and nearly 50% homology to the next 80 amino acids. Yeast RAS1 and RAS2 proteins are more homologous to each other, with about 90% homology for the first 180 positions. After this, at nearly the same position that the mammalian ras proteins begin to diverge from each other, the two yeast ras proteins diverge radically. The yeast ras proteins, like the proteins encoded by the mammalian genes, terminate with the sequence cysAAX, where A is an aliphatic amino acid. Thus the yeast ras proteins have the same overall structure and interrelationship as the family of mammalian ras proteins. The domains of divergence may correspond to functional domains of the ras proteins. Monoclonal antibody directed against mammalian ras proteins immunoprecipitates protein in yeast cells containing high copy numbers of the yeast RAS2 gene.     

Metabolic costs of amino acid and protein production in Escherichia coli

Escherichia coli is the most popular microorganism for the production of recombinant proteins and is gaining increasing importance for the production of low-molecular weight compounds such as amino acids. The metabolic cost associated with the production of amino acids and (recombinant) proteins from glucose, glycerol and acetate was determined using three different computational techniques to identify those amino acids that put the highest burden on the biosynthetic machinery of E. coli. Comparing the costs of individual amino acids, we find that methionine is the most expensive amino acid in terms of consumed mol of ATP per molecule produced, while leucine is the most expensive amino acid when taking into account the cellular abundances of amino acids. Moreover, we show that the biosynthesis of a large number of amino acids from glucose and particularly from glycerol provides a surplus of energy, which can be used to balance the high energetic cost of amino acid polymerization.     

Codon Usage in Plastid Genes Is Correlated with Context, Position Within the Gene, and Amino Acid Content

Highly expressed plastid genes display codon adaptation, which is defined as a bias toward a set of codons which are complementary to abundant tRNAs. This type of adaptation is similar to what is observed in highly expressed Escherichia coli genes and is probably the result of selection to increase translation efficiency. In the current work, the codon adaptation of plastid genes is studied with regard to three specific features that have been observed in E. coli and which may influence translation efficiency. These features are (1) a relatively low codon adaptation at the 5′ end of highly expressed genes, (2) an influence of neighboring codons on codon usage at a particular site (codon context), and (3) a correlation between the level of codon adaptation of a gene and its amino acid content. All three features are found in plastid genes. First, highly expressed plastid genes have a noticeable decrease in codon adaptation over the first 10–20 codons. Second, for the twofold degenerate NNY codon groups, highly expressed genes have an overall bias toward the NNC codon, but this is not observed when the 3′ neighboring base is a G. At these sites highly expressed genes are biased toward NNT instead of NNC. Third, plastid genes that have higher codon adaptations also tend to have an increased usage of amino acids with a high G + C content at the first two codon positions and GNN codons in particular. The correlation between codon adaptation and amino acid content exists separately for both cytosolic and membrane proteins and is not related to any obvious functional property. It is suggested that at certain sites selection discriminates between nonsynonymous codons based on translational, not functional, differences, with the result that the amino acid sequence of highly expressed proteins is partially influenced by selection for increased translation efficiency. Received: 21 July 1999 / Accepted: 5 November 1999     

Positive selection in phytotoxic protein-encoding genes of Botrytis species

Evolutionary patterns of sequence divergence were analyzed in genes from the fungal genus Botrytis (Ascomycota), encoding phytotoxic proteins homologous to a necrosis and ethylene-inducing protein from Fusarium oxysporum. Fragments of two paralogous genes (designated NEP1 and NEP2) were amplified from all known Botrytis species and sequenced. NEP1 sequences of two Botrytis species contain premature stop codons, indicating that they may be non-functional. Both paralogs of all species encode proteins with a remarkably similar predicted secondary structure, however, they contain different types of post-translational modification motifs, which are conserved across the genus. While both NEP genes are, overall, under purifying selection, we identified a number of amino acids under positive selection based on inference using maximum likelihood models. Positively selected amino acids in NEP1 were not under selection in corresponding positions in NEP2. The biological significance of positively selected residues and the role of NEP proteins in pathogenesis remain to be resolved.     

Amino Acid Metabolic Origin as an Evolutionary Influence on Protein Sequence in Yeast

The metabolic cycle of Saccharomyces cerevisiae consists of alternating oxidative (respiration) and reductive (glycolysis) energy-yielding reactions. The intracellular concentrations of amino acid precursors generated by these reactions oscillate accordingly, attaining maximal concentration during the middle of their respective yeast metabolic cycle phases. Typically, the amino acids themselves are most abundant at the end of their precursor’s phase. We show that this metabolic cycling has likely biased the amino acid composition of proteins across the S. cerevisiae genome. In particular, we observed that the metabolic source of amino acids is the single most important source of variation in the amino acid compositions of functionally related proteins and that this signal appears only in (facultative) organisms using both oxidative and reductive metabolism. Periodically expressed proteins are enriched for amino acids generated in the preceding phase of the metabolic cycle. Proteins expressed during the oxidative phase contain more glycolysis-derived amino acids, whereas proteins expressed during the reductive phase contain more respiration-derived amino acids. Rare amino acids (e.g., tryptophan) are greatly overrepresented or underrepresented, relative to the proteomic average, in periodically expressed proteins, whereas common amino acids vary by a few percent. Genome-wide, we infer that 20,000 to 60,000 residues have been modified by this previously unappreciated pressure. This trend is strongest in ancient proteins, suggesting that oscillating endogenous amino acid availability exerted genome-wide selective pressure on protein sequences across evolutionary time. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Benjamin L. de Bivort and Ethan O. Perlstein have contributed equally to this work.     

TAFA: a novel secreted family with conserved cysteine residues and restricted expression in the brain

We have discovered a family of small secreted proteins in Homo sapiens and Mus musculus using a novel database searching strategy. The family is composed of five highly homologous genes referred to as TAFA-1 to -5. The TAFA genes encode proteins of approximately 100 amino acids that contain conserved cysteine residues at fixed positions. TAFA-1 to -4 are more closely related to each other than to TAFA-5, in which a conserved motif including CC in TAFA-1 to -4 is not present. In H. sapiens, TAFA-3 has two isoforms formed by alternative splicing. Sequence homology analyses reveal that TAFA proteins appear distantly related to MIP-1alpha, a member of the CC-chemokine family. TAFA mRNAs are highly expressed in specific brain regions, with little expression seen in other tissues.     

Insight into the molecular evolution of two tropinone reductases

In tropane alkaloid biosynthesis, two tropinone reductases produce different stereoisomers from a common substrate, tropinone. The two enzymes share 64% of identical amino acids, and highly homologous proteins with variable substrate-binding residues have also been found in tropane alkaloid non-producing species. This exemplifies a simple evolutionary process that plants have taken to acquire a new secondary metabolic pathway.     

Cost-Minimization of Amino Acid Usage

The negative correlation between the frequencies of usage of amino acids and their biosynthetic cost suggests that organisms minimize costs of protein biosynthesis. Empirical results support that: (1) free-living organisms (Archaea, Bacteria, and Eucaryota) minimize the usage of heavy amino acids more than intracellular organisms (viruses, chloroplasts, and mitochondria), a result confirmed by comparing intracellular Bacteria with other Bacteria; (2) avoidance of amino acids with low impact on protein structure (Chou-Fasman indices) is greater than for those with equal molecular weight but greater structural impact: constraints on protein function limit cost-minimization; (3) amino acid weight minimization (WM) for a protein correlates positively with the protein's expression level and with its size; (4) preliminary results suggest that for different proteins, the evolutionary rate of amino acid replacements correlates negatively with WM in these proteins; (5) results suggest that WM decreases with genome-size; and (6) developmental rates correlate positively with WM (within primates and rodents), even after confounding factors were accounted for. Effects of biosynthetic cost-minimization at whole-organism levels vary with metabolic and ecological strategies. Biosynthetic cost-minimization is an adaptive hypothesis that yields a semi-mechanistic explanation for small differences in allele fitness.     

Bovine interferon alpha genes. Structure and expression

The bovine genome contains a gene family of interferon-alpha s (bIFN-alpha) that consists of at least five distinct members. Four of the bIFN-alpha genes isolated show a high degree of homology (97% in the nucleotide sequence and 93% in amino acid sequence). The overall homology in amino acid sequence of bIFN-alpha to human, murine, and rat IFNs-alpha is approximately 60%. Yet there are amino acid clusters (positions 28-41 and 118-146) which are highly conserved throughout the mammalian evolution and in which the overall homology can be as high as 86%. Within the C terminus conserved cluster there is a sequence containing 9 amino acids completely conserved in 16 mammalian IFNs-alpha and of these, 7 are also shared with a similar domain in some bacterial toxins, implying a common functional role for these domains. One of the genes, IFN-alpha C, was expressed in Escherichia coli. The purified bacterial IFN (specific activity, 2 X 10(8) units/mg) exhibited antiviral activity on bovine cells but no detectable activity was demonstrated on human and simian cells.     

Gene expression level shapes the amino acid usages in Prochlorococcus marinus MED4

Prochlorococcus species are the first example of free-living bacteria with reduced genome. Codon and amino acid usages bias of Prochlorococcus marinus MED4 was investigated using all protein coding genes having length greater than or equal to 100 amino acids. Correspondence analysis on relative synonymous codon usage (RSCU) values shows that there is no such influence of translational selection in shaping the codon usage variation among the genes in this organism. However, amino acid usages were markedly different between the highly and lowly expressed genes in this organism and in particular, GC rich amino acids were found to occur significantly higher in highly expressed genes than the lowly expressed genes. Comparative analysis of the homologous genes of Synechococcus sp. WH8102 and Prochlorococcus marinus MED4 shows that amino acids conservation in highly expressed genes is significantly higher than lowly expressed genes. Based on our results we concluded that conservation of GC rich amino acids in the highly expressed genes to its ancestor is the major source of variation in amino acid usages in the organism.     

Sequence of cDNAs encoding actin depolymerizing factor and cofilin of embryonic chicken skeletal muscle: two functionally distinct actin-regulatory proteins exhibit high structural homology.

Two actin-regulatory proteins of 19 and 20 kDa are involved in the regulation of actin assembly in developing chicken skeletal muscle. They are homologous with actin depolymerizing factor (ADF) and cofilin, a pH-dependent actin-modulating protein, which were originally discovered in chicken and mammalian brain, respectively. In this study, full-length cDNA clones were isolated by screening a lambda gt11 cDNA library constructed from poly(A+) RNA of embryonic chicken skeletal muscle with the antibodies specific for each protein, and their complete sequences were determined. The chicken cofilin cDNA encoded a protein of 166 amino acids, the sequence of which had over 80% identity with that of porcine brain cofilin. The amino acid sequence of the ADF was 165 amino acids and showed about 70% identity with either chicken or mammalian cofilin, in spite of the fact that ADF and cofilin are functionally distinct. Like chicken and mammalian cofilin, ADF contained a sequence similar to the nuclear transport signal sequence of SV40 large T antigen. ADF and cofilin shared a hexapeptide identical with the amino-terminal sequence of tropomyosin as well as the regions homologous to other actin-regulatory proteins, including depactin, gelsolin, and profilin. The overall nucleotide sequences and Southern blot analysis of genomic DNA, however, indicated that the two proteins were derived from different genes.