Construction of "small-intelligent" focused mutagenesis libraries using well-designed combinatorial degenerate primers

Site-saturation mutagenesis is a powerful tool for protein optimization due to its efficiency and simplicity. A degenerate codon NNN or NNS (K) is often used to encode the 20 standard amino acids, but this will produce redundant codons and cause uneven distribution of amino acids in the constructed library. Here we present a novel "small-intelligent" strategy to construct mutagenesis libraries that have a minimal gene library size without inherent amino acid biases, stop codons, or rare codons of Escherichia coli by coupling well-designed combinatorial degenerate primers with suitable PCR-based mutagenesis methods. The designed primer mixture contains exactly one codon per amino acid and thus allows the construction of small-intelligent mutagenesis libraries with one gene per protein. In addition, the software tool DC-Analyzer was developed to assist in primer design according to the user-defined randomization scheme for library construction. This small-intelligent strategy was successfully applied to the randomization of halohydrin dehalogenases with one or two randomized sites. With the help of DC-Analyzer, the strategy was proven to be as simple as NNS randomization and could serve as a general tool to efficiently randomize target genes at positions of interest.     

In vitro selection for sense codon suppression

The universal genetic code links the 20 naturally occurring amino acids to the 61 sense codons. Previously, the UAG amber stop codon (a nonsense codon) has been used as a blank in the code to insert natural and unnatural amino acids via nonsense suppression. We have developed a selection methodology to investigate whether the unnatural amino acid biocytin could be incorporated into an mRNA display library at sense codons. In these experiments we probed a single randomized NNN codon with a library of 16 orthogonal, biocytin-acylated tRNAs. In vitro selection for efficient incorporation of the unnatural amino acid resulted in templates containing the GUA codon at the randomized position. This sense suppression occurs via Watson-Crick pairing with similar efficiency to UAG-mediated nonsense suppression. These experiments suggest that sense codon suppression is a viable means to expand the chemical and functional diversity of the genetic code.     

Consequences of stop codon reassignment on protein evolution in ciliates with alternative genetic codes

Tetrahymena thermophila and Paramecium tetraurelia are ciliates that reassign TAA and TAG from stop codons to glutamine codons. Because of the lack of full genome sequences, few studies have concentrated on analyzing the effects of codon reassignment in protein evolution. We used the recently sequenced genome of these species to analyze the patterns of amino acid substitution in ciliates that reassign the code. We show that, as expected, the codon reassignment has a large impact on amino acid substitutions in closely related proteins; however, contrary to expectations, these effects also hold for very diverged proteins. Previous studies have used amino acid substitution data to calculate the minimization of the genetic code; our results show that because of the lasting influence of the code in the patterns of substitution, such studies are tautological. These different substitution patterns might affect alignment of ciliate proteins, as alignment programs use scoring matrices based on substitution patterns of organisms that use the standard code. We also show that glutamine is used more frequently in ciliates than in other species, as often as expected based on the presence of the 2 new reassigned codons, indicating that the frequencies of amino acids in proteomes is mostly determined by neutral processes based on their number of codons.     

Structure of the C-terminal end of the nascent peptide influences translation termination.

The efficiency of translation termination at NNN NNN UGA A stop codon contexts has been determined in Escherichia coli. No general effects are found which can be attributed directly to the mRNA sequences itself. Instead, termination is influenced primarily by the amino acids at the C-terminal end of the nascent peptide, which are specified by the two codons at the 5' side of UGA. For the penultimate amino acid (-2 location), charge and hydrophobicity are important. For the last amino acid (-1 location), alpha-helical, beta-strand and reverse turn propensities are determining factors. The van der Waals volume of the last amino acid can affect the relative efficiency of stop codon readthrough by the wild-type and suppressor forms of tRNA(Trp) (CAA). The influence of the -1 and -2 amino acids is cooperative. Accumulation of an mRNA degradation intermediate indicates mRNA protection by pausing ribosomes at contexts which give inefficient UGA termination. Highly expressed E.coli genes with the UGA A termination signal encode C-terminal amino acids which favour efficient termination. This restriction is not found for poorly expressed genes.     

The Selective Advantage of Synonymous Codon Usage Bias in Salmonella

The genetic code in mRNA is redundant, with 61 sense codons translated into 20 different amino acids. Individual amino acids are encoded by up to six different codons but within codon families some are used more frequently than others. This phenomenon is referred to as synonymous codon usage bias. The genomes of free-living unicellular organisms such as bacteria have an extreme codon usage bias and the degree of bias differs between genes within the same genome. The strong positive correlation between codon usage bias and gene expression levels in many microorganisms is attributed to selection for translational efficiency. However, this putative selective advantage has never been measured in bacteria and theoretical estimates vary widely. By systematically exchanging optimal codons for synonymous codons in the tuf genes we quantified the selective advantage of biased codon usage in highly expressed genes to be in the range 0.2–4.2 x 10⁻⁴ per codon per generation. These data quantify for the first time the potential for selection on synonymous codon choice to drive genome-wide sequence evolution in bacteria, and in particular to optimize the sequences of highly expressed genes. This quantification may have predictive applications in the design of synthetic genes and for heterologous gene expression in biotechnology.     

Quantitative codon optimisation of DNA libraries encoding sub-random peptides: design and characterisation of a novel library encoding transmembrane domain peptides

Codons for amino acids sharing similar chemical properties seem to cluster on the genetic codon table. Such a geographical distribution of the codons was exploited to create chemically synthesised DNA that encodes peptide libraries containing only a subset of the 20 natural amino acids. The frequency of each amino acid in the subset was further optimised by quantitatively manipulating the ratio of the four phosphoamidites during chemical synthesis of the libraries. Peptides encoded by such libraries show a reduced complexity and could be enriched in peptides of a desired property, which are thus more suitable when screening for functional peptides. Proof of concept for the codon-biased design of peptide libraries was shown by design, synthesis, and characterisation of a transmembrane peptide library that contains >80% transmembrane peptides, representing a 160-fold enrichment compared with a fully randomised library.     

The genetic code--40 years on

The genetic code discovered 40 years ago, consists of 64 triplets (codons) of nucleotides. The genetic code is almost universal. The same codons are assigned to the same amino acids and to the same START and STOP signals in the vast majority of genes in animals, plants, and microorganisms. Each codon encodes for one of the 20 amino acids used in the synthesis of proteins. That produces some redundancy in the code and most of the amino acids being encoded by more than one codon. The two cases have been found where selenocysteine or pyrrolysine, that are not one of the standard 20 is inserted by a tRNA into the growing polypeptide.     

When second best is good enough: another probabilistic look at saturation mutagenesis

We developed new criteria for determining the library size in a saturation mutagenesis experiment. When the number of all possible distinct variants is large, any of the top-performing variants (e.g., any of the top three) is likely to meet the design requirements, so the probability that the library contains at least one of them is a sensible criterion for determining the library size. By using a criterion of this type, one may significantly reduce the library size and thus save costs and labor while minimally compromising the quality of the best variant discovered. We present the probabilistic tools underlying these criteria and use them to compare the efficiencies of four randomization schemes: NNN, which uses all 64 codons; NNB, which uses 48 codons; NNK, which uses 32 codons; and MAX, which assigns equal probabilities to each of the 20 amino acids. MAX was found to be the most efficient randomization scheme and NNN the least efficient. TopLib, a computer program for carrying out the related calculations, is available through a user-friendly Web server.     

三联体密码子在mRNA二级结构中的分布

对编码成熟肽的ｍＲＮＡ二级结构的分析显示,每个密码子在ｍＲＮＡ二级结构中的位置有一定的倾向性,这种倾向性似乎与相应氨基酸的构象性质相一致。大多数编码疏水氨基酸的密码子位于ｍＲＮＡ二级结构中较稳定的茎区;反之,大多数编码亲水氨基酸的密码子位于柔性的环区。这个结果支持了最近得到的关于ｍＲＮＡ与蛋白质之间存在丰三维结构信息传递的结论。     

Configuron dependence on translation of specific condon pairs. I. Helical regions of human alpha and beta globins

Homologous proteins, which possess similar shapes, functions, and amino acid sequences, are encoded by homologous messenger ribonucleic acids whose codon sequences tend to be similar. It is proposed that helical configurons are generated when certain pairs of contigous codons are translated, and that non-helical configurons appear when other specific pairs of codons are read off. The resulting sequence of configurons comprises the polyconfiguron, which forms the native structure of the protein.     

A Model of Proto-Anti-Codon RNA Enzymes Requiring l-Amino Acid Homochirality

All living organisms encode the 20 natural amino acid units of polypeptides using a universal scheme of triplet nucleotide "codons". Disparate features of this codon scheme are potentially informative of early molecular evolution: (i) the absence of any codons for D-amino acids; (ii) the odd combination of alternate codon patterns for some amino acids; (iii) the confinement of synonymous positions to a codon's third nucleotide; (iv) the use of 20 specific amino acids rather than a number closer to the full coding potential of 64; and (v) the evolutionary relationship of patterns in stop codons to amino acid codons. Here I propose a model for an ancestral proto-anti-codon RNA (pacRNA) auto-aminoacylation system and show that pacRNAs would naturally manifest features of the codon table. I show that pacRNAs could implement all the steps for auto-aminoacylation: amino acid coordination, intermediate activation of the amino acid by the 5'-end of the pacRNA, and 3'-aminoacylation of the pacRNA. The anti-codon cradles of pacRNAs would have been able to recognize and coordinate only a small number of L-amino acids via hydrogen bonding. A need for proper spatial coordination would have limited the number of chargeable amino acids for all anti-codon sequences, in addition to making some anti-codon sequences unsuitable. Thus, the pacRNA model implies that the idiosyncrasies of the anti-codon table and L-amino acid homochirality co-evolved during a single evolutionary period. These results further imply that early life consisted of an aminoacylated RNA world with a richer enzymatic potential than ribonucleotides alone.     

Second codon positions of genes and the secondary structures of proteins. Relationships and implications for the origin of the genetic code

The nucleotide frequencies in the second codon positions of genes are remarkably different for the coding regions that correspond to different secondary structures in the encoded proteins, namely, helix, beta-strand and aperiodic structures. Indeed, hydrophobic and hydrophilic amino acids are encoded by codons having U or A, respectively, in their second position. Moreover, the beta-strand structure is strongly hydrophobic, while aperiodic structures contain more hydrophilic amino acids. The relationship between nucleotide frequencies and protein secondary structures is associated not only with the physico-chemical properties of these structures but also with the organisation of the genetic code. In fact, this organisation seems to have evolved so as to preserve the secondary structures of proteins by preventing deleterious amino acid substitutions that could modify the physico-chemical properties required for an optimal structure.     

Codon usage in higher plants, green algae, and cyanobacteria

Codon usage is the selective and nonrandom use of synonymous codons by an organism to encode the amino acids in the genes for its proteins. During the last few years, a large number of plant genes have been cloned and sequenced, which now permits a meaningful comparison of codon usage in higher plants, algae, and cyanobacteria. For the nuclear and organellar genes of these organisms, a small set of preferred codons are used for encoding proteins. Codon usage is different for each genome type with the variation mainly occurring in choices between codons ending in cytidine (C) or guanosine (G) versus those ending in adenosine (A) or uridine (U). For organellar genomes, chloroplastic and mitochrondrial proteins are encoded mainly with codons ending in A or U. In most cyanobacteria and the nuclei of green algae, proteins are encoded preferentially with codons ending in C or G. Although only a few nuclear genes of higher plants have been sequenced, a clear distinction between Magnoliopsida (dicot) and Liliopsida (monocot) codon usage is evident. Dicot genes use a set of 44 preferred codons with a slight preference for codons ending in A or U. Monocot codon usage is more restricted with an average of 38 codons preferred, which are predominantly those ending in C or G. But two classes of genes can be recognized in monocots. One set of monocot genes uses codons similar to those in dicots, while the other genes are highly biased toward codons ending in C or G with a pattern similar to nuclear genes of green algae. Codon usage is discussed in relation to evolution of plants and prospects for intergenic transfer of particular genes.     

Some theoretical aspects of reprogramming the standard genetic code

Reprogramming of the standard genetic code to include non-canonical amino acids (ncAAs) opens new prospects for medicine, industry, and biotechnology. There are several methods of code engineering, which allow us for storing new genetic information in DNA sequences and producing proteins with new properties. Here, we provided a theoretical background for the optimal genetic code expansion, which may find application in the experimental design of the genetic code. We assumed that the expanded genetic code includes both canonical and non-canonical information stored in 64 classical codons. What is more, the new coding system is robust to point mutations and minimizes the possibility of reversion from the new to old information. In order to find such codes, we applied graph theory to analyze the properties of optimal codon sets. We presented the formal procedure in finding the optimal codes with various number of vacant codons that could be assigned to new amino acids. Finally, we discussed the optimal number of the newly incorporated ncAAs and also the optimal size of codon groups that can be assigned to ncAAs.     

Codon-based mutagenesis using dimer-phosphoramidites.

A new approach for the synthesis of randomized DNA sequences containing the 20 codons corresponding to all natural amino acids is described. The strategy is based on the use of dinucleotide phosphoramidite building blocks within a resin-splitting procedure. Through this protocol, a minimal number of seven dimers is sufficient to encode all 20 natural amino acids. This synthesis procedure is extremely flexible and allows codon usage from different hosts to be accommodated.     

Codon usage bias in prokaryotic pyrimidine-ending codons is associated with the degeneracy of the encoded amino acids

Synonymous codons are unevenly distributed among genes, a phenomenon termed codon usage bias. Understanding the patterns of codon bias and the forces shaping them is a major step towards elucidating the adaptive advantage codon choice can confer at the level of individual genes and organisms. Here, we perform a large-scale analysis to assess codon usage bias pattern of pyrimidine-ending codons in highly expressed genes in prokaryotes. We find a bias pattern linked to the degeneracy of the encoded amino acid. Specifically, we show that codon-pairs that encode two- and three-fold degenerate amino acids are biased towards the C-ending codon while codons encoding four-fold degenerate amino acids are biased towards the U-ending codon. This codon usage pattern is widespread in prokaryotes, and its strength is correlated with translational selection both within and between organisms. We show that this bias is associated with an improved correspondence with the tRNA pool, avoidance of mis-incorporation errors during translation and moderate stability of codon-anticodon interaction, all consistent with more efficient translation.     

Comparison of the frequency of functional SH3 domains with different limited sets of amino acids using mRNA display

Although modern proteins consist of 20 different amino acids, it has been proposed that primordial proteins consisted of a small set of amino acids, and additional amino acids have gradually been recruited into the genetic code. This hypothesis has recently been supported by comparative genome sequence analysis, but no direct experimental approach has been reported. Here, we utilized a novel experimental approach to test a hypothesis that native-like globular proteins might be easily simplified by a set of putative primitive amino acids with retention of its structure and function than by a set of putative new amino acids. We performed in vitro selection of a functional SH3 domain as a model from partially randomized libraries with different sets of amino acids using mRNA display. Consequently, a library rich in putative primitive amino acids included a larger number of functional SH3 sequences than a library rich in putative new amino acids. Further, the functional SH3 sequences were enriched from the primitive library slightly earlier than from a randomized library with the full set of amino acids, while the function and structure of the selected SH3 proteins with the primitive alphabet were comparable with those from the 20 amino acid alphabet. Application of this approach to various combinations of codons in protein sequences may be useful not only for clarifying the precise order of the amino acid expansion in the early stages of protein evolution but also for efficiently creating novel functional proteins in the laboratory.     

A hardware interpretation of the evolution of the genetic code

A quantitative rationale for the evolution of the genetic code is developed considering the principle of minimal hardware. This principle defines an optimal code as one that minimizes for a given amount of information encoded, the product of the number of physical devices used by the average complexity of each device. By identifying the number of different amino acids, number of nucleotide positions per codon and number of base types that can occupy each such position with, respectively, the amount of information, number of devices and the complexity, we show that optimal codes occur for 3, 7 and 20 amino acids with codons having a single, two and three base positions per codon, respectively. The advantage of a code of exactly 4 symbols is deduced, as well as a plausible evolutionary pathway from a code of doublets to triplets. The present day code of 20 amino acids encoded by 64 codons is shown to be the most optimal in an absolute sense. Using a tetraplet code further evolution to a code in which there would be 55 amino acids is in principle possible, but such a code would deviate slightly more than the present day code from the minimal hardware configuration. The change from a triplet code to a tetraplet code would occur at about 32 amino acids. Our conclusions are independent of, but consistent with, the observed physico-chemical properties of the amino acids and codon structures. These correlations could have evolved within the constrains imposed by the minimal hardware principle.     

Isolation and expression of two genes encoding eukaryotic release factor 1 from Paramecium tetraurelia

Paramecium tetraurelia, like some other ciliate species, uses an alternative nuclear genetic code where UAA and UAG are translated as glutamine and UGA is the only stop codon. It has been postulated that the use of stop codons as sense codons is dependent on the presence of specific tRNAs and on modification of eukaryotic release factor one (eRF1), a factor involved in stop codon recognition during translation termination. We describe here the isolation and characterisation of two genes, eRF1-a and eRF1 b, coding for eRF1 in P. tetraurelia. The two genes are very similar, both in genomic organization and in sequence, and might result from a recent duplication event. The two coding sequences are 1,314 nucleotides long, and encode two putative proteins of 437 amino acids with 98.5% identity. Interestingly, when compared with the eRF1 sequences either of ciliates having the same variant genetic code, or of other eukaryotes, the eRF1 of P. tetraurelia exhibits significant differences in the N-terminal region, which is thought to interact with stop codons. We discuss here the consequences of these changes in the light of recent models proposed to explain the mechanism of stop codon recognition in eukaryotes. Besides, analysis of the expression of the two genes by Northern blotting and primer extension reveals that these genes exhibit a differential expression during vegetative growth and autogamy.     

On the Evolution of Redundancy in Genetic Codes

We simulate a deterministic population genetic model for the coevolution of genetic codes and protein-coding genes. We use very simple assumptions about translation, mutation, and protein fitness to calculate mutation-selection equilibria of codon frequencies and fitness in a large asexual population with a given genetic code. We then compute the fitnesses of altered genetic codes that compete to invade the population by translating its genes with higher fitness. Codes and genes coevolve in a succession of stages, alternating between genetic equilibration and code invasion, from an initial wholly ambiguous coding state to a diversified frozen coding state. Our simulations almost always resulted in partially redundant frozen genetic codes. Also, the range of simulated physicochemical properties among encoded amino acids in frozen codes was always less than maximal. These results did not require the assumption of historical constraints on the number and type of amino acids available to codes nor on the complexity of proteins, stereochemical constraints on the translational apparatus, nor mechanistic constraints on genetic code change. Both the extent and timing of amino-acid diversification in genetic codes were strongly affected by the message mutation rate and strength of missense selection. Our results suggest that various omnipresent phenomena that distribute codons over sites with different selective requirements—such as the persistence of nonsynonymous mutations at equilibrium, the positive selection of the same codon in different types of sites, and translational ambiguity—predispose the evolution of redundancy and of reduced amino acid diversity in genetic codes. Received: 21 December 2000 / Accepted: 12 March 2001