The role of protein associated amino acid precursor molecules in the organization of genetic codons

A better understanding of the origin and organization of genetic codons is possible based on the metabolic relatedness of amino acids. Amino acids with similar codons (anticodons) usually have the same or similar precursor molecule, even if the amino acids are not related physico-chemically. These observations suggest, that amino acid precursor molecules and enzymes responsible for the synthesis of amino acids "must have seen" the protein synthesis machinery, and played a fundamental role in the codon (anticodon) organization.     

Four-base codon/anticodon strategy and non-enzymatic aminoacylation for protein engineering with non-natural amino acids

Techniques for position-specific incorporation of non-natural amino acids in an in vitro protein synthesizing system are described. First, a PNA-assisted non-enzymatic tRNA aminoacylation with a variety of natural and non-natural amino acids is described. With this technique, one can aminoacylate a specific tRNA simply by adding a preformed amino acid activated ester-PNA conjugate into an in vitro protein biosynthesizing system. Second, the genetic code is expanded by introducing 4-base codons that can be exclusively translated to non-natural amino acids. The most advantageous point of the 4-base codon strategy is to introduce multiple amino acids into specific positions in single proteins by using mutually orthogonal 4-base codons and orthogonal tRNAs. An easy and quick method for preparation of tRNAs possessing 4-base anticodons is also described. Combination of the non-enzymatic aminoacylation and the 4-base codon/anticodon strategy gives an easy and widely applicable technique for incorporating a variety of non-natural amino acids into proteins in vitro.     

Introduction of specialty functions by the position-specific incorporation of nonnatural amino acids into proteins through four-base codon/anticodon pairs

Position-specific incorporation of nonnatural amino acids into proteins (nonnatural mutagenesis) via an in vitro protein synthesizing system was applied to incorporate a variety of amino acids carrying specialty side groups. A list of nonnatural amino acids thus far successfully incorporated through in vitro translation systems is presented. The position of nonnatural amino acid incorporation was directed by four-base codon/anticodon pairs such as CGGG/CCCG and AGGU/ACCU. The four-base codon strategy was more efficient than the amber codon strategy and could incorporate multiple nonnatural amino acids into single proteins. This multiple mutagenesis will find wide applications, especially in building paths of electron transfer on proteins. The extension of translation systems by the introduction of nonnatural amino acids, four-base codon/anticodon pairs, orthogonal tRNAs, and artificial aminoacyl tRNA synthetases, is a promising approach towards the creation of "synthetic microorganisms" with specialty functions.     

Origin of life: A hypothesis for the origin of adaptor-mediated ordered synthesis of proteins and an explanation for the choice of terminating codons in the genetic code

Life can be defined as a system of self-sustained chemical processes springing from the ordered synthesis of proteins directed by nucleic acids. To the notoriously difficult problem of the origin of this basic process of nucleic acid-directed protein synthesis, we give a solution of molecular interactions between pentanucleotides and amino acids. A particular conformation of a pentanucleotide forms a double sided template, with its ‘inside’ capable of nestling an amino acid while the ‘outside’ acts as an adaptor to a ‘codon’ triplet on long-chain nucleic acids. This serves as a primitive decoding system. An important aspect of our postulate is that a dynamic interaction is triggered, by this decoding system, through which amino acids are brought to juxtaposition facilitating peptide bond formation. Almost all the important and unique features of contemporary protein-synthesizing machinery are seen to be a direct and natural consequence of our postulate. The emergence of the termination codons also fits in, as a natural consequence of this molecular mechanism.     

Effects of release factor 1 on in vitro protein translation and the elaboration of proteins containing unnatural amino acids.

An in vitro protein synthesizing system was modified to facilitate the improved, site-specific incorporation of unnatural amino acids into proteins via readthrough of mRNA nonsense (UAG) codons by chemically misacylated suppressor tRNAs. The modified system included an S-30 extract derived from Escherichia coli that expresses a temperature-sensitive variant of E. coli release factor 1 (RF1). Mild heat treatment of the S-30 extract partially deactivated RF1 and improved UAG codon readthrough by as much as 11-fold, as demonstrated by the incorporation of unnatural amino acids into positions 25 and 125 of HIV-1 protease and positions 10 and 22 of E. coli dihydrofolate reductase. The increases in yields were the greatest for those amino acids normally incorporated poorly in the in vitro protein synthesizing system, thus significantly enhancing the repertoire of modified amino acids that can be incorporated into the proteins of interest. The substantial increase in mutant protein yields over those obtained with an S-30 extract derived from an RF1 proficient E. coli strain is proposed to result from a relaxed stringency of termination by RF1 at the stop codon (UAG). When RF1 levels were depleted further, the intrinsic rate of DHFR synthesis increased, consistent with the possibility that RF1 competes not only at stop codons but also at other mRNA codons during peptide elongation. It thus seems possible that in addition to its currently accepted role as a protein factor involved in peptide termination, RF1 is also involved in functions that control the rate at which protein synthesis proceeds.     

Experimental studies on the origin of the genetic code and the process of protein synthesis: A review update

This article is an update of our earlier review (Lacey and Mullins, 1983) in this journal on the origin of the genetic code and the process of protein synthesis. It is our intent to discuss only experimental evidence published since then although there is the necessity to mention the old enough to place the new in context. We do not include theoretical nor hypothetical treatments of the code or protein synthesis. Relevant data regarding the evolution of tRNAs and the recognition of tRNAs by aminoacyl-tRNA-synthetases are discussed. Our present belief is that the code arose based on a core of early assignments which were made on a physico-chemical and anticodonic basis and this was expanded with new assignments later. These late assignments do not necessarily show an amino acid-anticodon relatedness. In spite of the fact that most data suggest a code origin based on amino acid-anticodon relationships, some new data suggesting preferential binding of Arg to its codons are discussed. While information regarding coding is not increasing very rapidly, information regarding the basic chemistry of the process of protein synthesis has increased significantly, principally relating to aminoacylation of mono- and polyribonucleotides. Included in those studies are several which show stereoselective reactions of L-amino acids with nucleotides having D-sugars. Hydrophobic interactions definitely play a role in the preferences which have been observed.     

A biophysical basis for the emergence of the genetic code in protocells

The origin of the genetic code is an abiding mystery in biology. Hints of a ‘code within the codons’ suggest biophysical interactions, but these patterns have resisted interpretation. Here, we present a new framework, grounded in the autotrophic growth of protocells from CO₂ and H₂. Recent work suggests that the universal core of metabolism recapitulates a thermodynamically favoured protometabolism right up to nucleotide synthesis. Considering the genetic code in relation to an extended protometabolism allows us to predict most codon assignments. We show that the first letter of the codon corresponds to the distance from CO₂ fixation, with amino acids encoded by the purines (G followed by A) being closest to CO₂ fixation. These associations suggest a purine-rich early metabolism with a restricted pool of amino acids. The second position of the anticodon corresponds to the hydrophobicity of the amino acid encoded. We combine multiple measures of hydrophobicity to show that this correlation holds strongly for early amino acids but is weaker for later species. Finally, we demonstrate that redundancy at the third position is not randomly distributed around the code: non-redundant amino acids can be assigned based on size, specifically length. We attribute this to additional stereochemical interactions at the anticodon. These rules imply an iterative expansion of the genetic code over time with codon assignments depending on both distance from CO₂ and biophysical interactions between nucleotide sequences and amino acids. In this way the earliest RNA polymers could produce non-random peptide sequences with selectable functions in autotrophic protocells.     

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.     

Multiple incorporation of non-natural amino acids into a single protein using tRNAs with non-standard structures

The ability to introduce non-natural amino acids into proteins opens up new vistas for the study of protein structure and function. This approach requires suppressor tRNAs that deliver the non-natural amino acid to a ribosome associated with an mRNA containing an expanded codon. The suppressor tRNAs must be absolutely protected from aminoacylation by any of the aminoacyl-tRNA synthetases in the protein synthesizing system, or a natural amino acid will be incorporated instead of the non-natural amino acid. Here, we found that some tRNAs with non-standard structures could work as efficient four-base suppressors fulfilling the above orthogonal conditions. Using these tRNAs, we successfully demonstrated incorporation of three different non-natural amino acids into a single protein.     

非天然氨基酸正交翻译技术：一种新型基因工程活病毒疫苗研发技术

非天然氨基酸正交翻译技术利用外源的非天然氨基酸氨酰tRNA合成酶(aaRS)基因和对应的tRNA基因构建非天然氨基酸正交翻译系统(Orthogonal translation system)。该正交翻译系统能利用终止密码子在蛋白翻译过程中将非天然氨基酸定点插入目标多肽链中。该技术不但是一种新的蛋白质生化研究工具,在新型基因工程病毒疫苗研究中更具有划时代的意义。利用人为构建的具有非天然氨基酸正交翻译系统的转基因细胞,通过在病毒复制的关键基因中引入提前终止密码子构建的突变病毒,在添加非天然氨基酸的情况下该基因仍能完整表达从而完成病毒的复制和传代,但该突变病毒在正常细胞(无非天然氨基酸正交翻译系统的宿主细胞)中因复制关键基因不能完整表达而无法复制传代,因而是一种复制缺陷型病毒。这种复制缺陷型病毒用作疫苗时兼具了减毒活疫苗免疫效果良好与灭活疫苗安全性高的优点,是一种较为理想的活病毒疫苗。文中简要综述了非天然氨基酸正交翻译技术在新型复制缺陷活病毒疫苗研究中的应用及其前景。     

Extension of biochemical functions by the introduction of nonnatural amino acids and artificial nucleic acid analogs.

Extension of biochemical functions has been attempted by introducing nonatural amino acids and artificial nucleic acid analogs. Nonnatural amino acids have been linked to tRNAs and the amino-acylated tRNAs were added to E. coli in vitro protein synthesizing system to produce nonnatural mutant proteins. The positions of the nonnatural amino acids have been assigned by the 4-base codons, like CGGG and AGGU. The extended codons have been introduced at a specific position or at random positions on a DNA. In the latter case, a DNA library that contains a single 4-base codon at random positions can be obtained. The combination of these new techniques opens a way to the introduction of artificial functions to biochemical systems.     

In vivo engineering of proteins with nitrogen-containing tryptophan analogs

Recently, it has become possible to reprogram the protein synthesis machinery such that numerous noncanonical amino acids can be translated into target sequences yielding tailor-made proteins. The canonical amino acid tryptophan (Trp) encoded by a single nucleotide triplet (UGG) is a particularly interesting target for protein engineering and design. Trp-residues can be substituted with a variety of analogs and surrogates generated biosynthetically or by organic chemistry. Among them, nitrogen-containing tryptophan analogs occupy a central position, as they have distinct chemical properties in comparison with aliphatic amines and imines. They resemble purine bases of DNA and share their capacity for pH-sensitive intramolecular charge transfer. These special properties of the analogs can be directly transmitted into related protein structures via in vivo ribosome-mediated translation. Proteins expressed in this way are further endowed with unique properties like new spectral, altered redox and titration features or might serve as useful biomaterials. We present and discuss current works and future developments in protein engineering with nitrogen-containing tryptophan analogs and related compounds as well as their relevance for academic and applicative research.The term noncanonical amino acid refers to an amino acid that does not belong, in contrast to a canonical amino acid, to the genetically encoded, proteinogenic amino acids. The term analog defines a strict isosteric exchange of a canonical/noncanonical amino acid (e.g., tryptophan/azatryptophan), while the term surrogate defines a nonisosteric change (e.g., tryptophan/azulene). Mutant denotes a protein in which the wild-type sequence was changed by site-directed mutagenesis (codon manipulation on the DNA level) within the repertoire of the standard amino acids. Variant denotes a protein in which one or more canonical amino acids derived from a wild-type or a mutant sequence were replaced by a noncanonical one (expanded amino acid repertoire, codon reassignment on the protein translation level).     

The mitochondrial pool of free amino acids reflects the composition of mitochondrial DNA-encoded proteins: indication of a post- translational quality control for protein synthesis

Mitochondria can synthesize a limited number of proteins encoded by mtDNA (mitochondrial DNA) by using their own biosynthetic machinery, whereas most of the proteins in mitochondria are imported from the cytosol. It could be hypothesized that the mitochondrial pool of amino acids follows the frequency of amino acids in mtDNA-encoded proteins or, alternatively, that the profile is the result of the participation of amino acids in pathways other than protein synthesis (e.g. haem biosynthesis and aminotransferase reactions). These hypotheses were tested by evaluating the pool of free amino acids and derivatives in highly-coupled purified liver mitochondria obtained from rats fed on a nutritionally adequate diet for growth. Our results indicated that the pool mainly reflects the amino acid composition of mtDNA-encoded proteins, suggesting that there is a post-translational control of protein synthesis. This conclusion was supported by the following findings: (i) correlation between the concentration of free amino acids in the matrix and the frequency of abundance of amino acids in mtDNA-encoded proteins; (ii) the similar ratios of essential-to-non-essential amino acids in mtDNA-encoded proteins and the mitochondrial pool of amino acids; and (iii), lack of a correlation between codon usage or tRNA levels and amino-acid concentrations. Quantitative information on the mammalian mitochondrial content of amino acids, such as that presented in the present study, along with functional studies, will help us to better understand the pathogenesis of mitochondrial diseases or the biochemical implications in mitochondrial metabolism.     

Stereochemical origins of the genetic code

The origin of the genetic code may be attributed to a postulated prebiological stereochemistry in which amino acid dimers, the trans -R,R'-diketopiperazines, interacted with prototype codon and anticodon nucleotide sequences. An intricately coupled stereochemistry is formulated which displays a binary logic for amino acid-codon recognition. It is shown that the diketopiperazine ring system can be inserted between any terminal pair of base paired nucleotides in a codon-anticodon structure with exact registration of complementary hydrogen bonding functional groups. This yields a codon-dimer-anticodon structure in which each amino acid residue is projected towards and interacts with a particular sequence of vicinal nucleotides on either codon or anticodon. The projection direction and the sequence of nucleotides encountered is a strongly coupled function of the choice of codon terminal nucleotide and the handedness of the amino acid. The reciprocal chemical nature of the complementary base pairs drives the selection of dimers containing quite dissimilar and chirally opposed amino acids. Application of the stereochemical model to the in vivo system leads to a general correlation for amino acid-codon assignments. The genetic code is restated in terms of the dimers selected. The profound symmetry of the code is elucidated and this proves useful for correlative and predictive purposes.     

A single aromatic core mutation converts a designed “primitive” protein from halophile to mesophile folding

The halophile environment has a number of compelling aspects with regard to the origin of structured polypeptides (i.e., proteogenesis) and, instead of a curious niche that living systems adapted into, the halophile environment is emerging as a candidate “cradle” for proteogenesis. In this viewpoint, a subsequent halophile‐to‐mesophile transition was a key step in early evolution. Several lines of evidence indicate that aromatic amino acids were a late addition to the codon table and not part of the original “prebiotic” set comprising the earliest polypeptides. We test the hypothesis that the availability of aromatic amino acids could facilitate a halophile‐to‐mesophile transition by hydrophobic core‐packing enhancement. The effects of aromatic amino acid substitutions were evaluated in the core of a “primitive” designed protein enriched for the 10 prebiotic amino acids (A,D,E,G,I,L,P,S,T,V)—having an exclusively prebiotic core and requiring halophilic conditions for folding. The results indicate that a single aromatic amino acid substitution is capable of eliminating the requirement of halophile conditions for folding of a “primitive” polypeptide. Thus, the availability of aromatic amino acids could have facilitated a critical halophile‐to‐mesophile protein folding adaptation—identifying a selective advantage for the incorporation of aromatic amino acids into the codon table.     

Evolution of selenocysteine-containing proteins: significance of identification and functional characterization of selenoproteins

In the genetic code, UGA serves as either a signal for termination or a codon for selenocysteine (Sec). Sec rarely occurs in protein and is different from other amino acids in that much of the biosynthetic machinery governing its incorporation into protein is unique to this amino acid. Sec-containing proteins have diverse functions and lack a common amino acid motif or consensus sequence. Sec has previously been considered to be a relic of the primordial genetic code that was counter-selected by the presence of oxygen in the atmosphere. In the present report, it is proposed that Sec was added to the already existing genetic code and its use has accumulated during evolution of eukaryotes culminating in vertebrates. The more recently evolved selenoproteins appear to take advantage of unique redox properties of Sec that are superior to those of Cys for specific biological functions. Further understanding of the evolution of selenoproteins as well as biological properties and biomedical applications of the trace element selenium requires identification and functional characterization of all mammalian selenoproteins.     

Natural self-organization of polynucleotides and polypeptides in protobiogenesis: Appearance of a protohypercycle

Basic thermal polyamino acids or proteinoids have been reported to be catalytic for both self-instructing polymerization of amino acids and internucleotide synthesis. We show theoretically that a complex suspension of thermal proteinoids, free amino acids, nucleotides and ATP as an energy source can exhibit an evolutionary character. The suspension can produce a prototype of Eigen's hypercycle, or protohypercycle, for which translation proceeds from amino acid to nucleotide. The protohypercycle is suggested to be an evolutionary precursor of the hypercycle, in which translation is from nucleotide to amino acid. The possibility that the Fox-Nakashima microsphere containing both lysine-rich and acidic proteinoids may work as a model of a protohypercycle is considered.     

TTG serves as an initiation codon for the ribosomal protein MvaS7 from the archaeon Methanococcus vannielii.

The ribosomal protein MvaS7 from the methanogenic archaeon Methanococcus vannielii is a protein of 188 amino acids, i.e., it is 42 amino acids longer than previously suggested. The triplet TTG serves as a start codon. The methanogenic translation initiation region that includes the rare TTG start codon is recognized in Escherichia coli.     

Amino Acid Exchangeability and the Adaptive Code Hypothesis

Since the genetic code first was determined, many have claimed that it is organized adaptively, so as to assign similar codons to similar amino acids. This claim has proved difficult to establish due to the absence of relevant comparative data on alternative primordial codes and of objective measures of amino acid exchangeability. Here we use a recently developed measure of exchangeability to evaluate a null hypothesis and two alternative hypotheses about the adaptiveness of the genetic code. The null hypothesis that there is no tendency for exchangeable amino acids to be assigned to similar codons can be excluded here as expected from earlier work. The first alternative hypothesis is that any such correlation between codon distance and amino acid distance is due to incremental mechanisms of code evolution, and not to adaptation to reduce deleterious effects of future mutations. More specifically, new codon assignments that occur by ambiguity reduction or by codon capture will tend to give rise to correlations, whether due to the condition of amino acid ambiguity, or to the condition of similarity between a new tRNA synthetase (or tRNA) and its parent. The second alternative hypothesis, the adaptive hypothesis, then may be defined as an excess relative to what may be expected given the incremental nature of evolution, reflecting true adaptation for robustness rather than an incidental effect. The results reported here indicate that most of the nonrandomness in the amino acids to codon assignments can be explained by incremental code evolution, with a small residue of orderliness that may reflect code adaptation.     

Reassignment of sense codons in vivo

The genetic code maps one or more of the 61 sense codons onto a set of 20 canonical amino acids. Reassignment of sense codons to non-canonical amino acids in model organisms such as Escherichia coli has been achieved through manipulation of the cellular protein synthesis machinery. Specifically, control of amino acid pools, coupled with engineering of the aminoacyl-tRNA synthetase activity of the host, has enabled assignment of sense codons to a wide variety of non-canonical amino acids under conditions routinely used for expression of recombinant proteins. Codon reassignment is leading to important advances in protein engineering and bioorganic chemistry. Here we summarize some of those advances, and provide detailed protocols for codon reassignment.