Accurate translation of the genetic code depends on tRNA modified nucleosides

Transfer RNA molecules translate the genetic code by recognizing cognate mRNA codons during protein synthesis. The anticodon wobble at position 34 and the nucleotide immediately 3' to the anticodon triplet at position 37 display a large diversity of modified nucleosides in the tRNAs of all organisms. We show that tRNA species translating 2-fold degenerate codons require a modified U(34) to enable recognition of their cognate codons ending in A or G but restrict reading of noncognate or near-cognate codons ending in U and C that specify a different amino acid. In particular, the nucleoside modifications 2-thiouridine at position 34 (s(2)U(34)), 5-methylaminomethyluridine at position 34 (mnm(5)U(34)), and 6-threonylcarbamoyladenosine at position 37 (t(6)A(37)) were essential for Watson-Crick (AAA) and wobble (AAG) cognate codon recognition by tRNA(UUU)(Lys) at the ribosomal aminoacyl and peptidyl sites but did not enable the recognition of the asparagine codons (AAU and AAC). We conclude that modified nucleosides evolved to modulate an anticodon domain structure necessary for many tRNA species to accurately translate the genetic code.     

Unassigned Codons, Nonsense Suppression, and Anticodon Modifications in the Evolution of the Genetic Code

The origin of the genetic code is a central open problem regarding the early evolution of life. Here, we consider two undeveloped but important aspects of possible scenarios for the evolutionary pathway of the translation machinery: the role of unassigned codons in early stages of the code and the incorporation of tRNA anticodon modifications. As the first codons started to encode amino acids, the translation machinery likely was faced with a large number of unassigned codons. Current molecular scenarios for the evolution of the code usually assume the very rapid assignment of all codons before all 20 amino acids became encoded. We show that the phenomenon of nonsense suppression as observed in current organisms allows for a scenario in which many unassigned codons persisted throughout most of the evolutionary development of the code. In addition, we demonstrate that incorporation of anticodon modifications at a late stage is feasible. The wobble rules allow a set of 20 tRNAs fully lacking anticodon modifications to encode all 20 canonical amino acids. These observations have implications for the biochemical plausibility of early stages in the evolution of the genetic code predating tRNA anticodon modifications and allow for effective translation by a relatively small and simple early tRNA set.     

Forced Ambiguity of the Leucine Codons for Multiple-Site-Specific Incorporation of a Noncanonical Amino Acid

Multiple-site-specific incorporation of a noncanonical amino acid into a recombinant protein would be a very useful technique to generate multiple chemical handles for bioconjugation and multivalent binding sites for the enhanced interaction. Previously combination of a mutant yeast phenylalanyl-tRNA synthetase variant and the yeast phenylalanyl-tRNA containing the AAA anticodon was used to incorporate a noncanonical amino acid into multiple UUU phenylalanine (Phe) codons in a site-specific manner. However, due to the less selective codon recognition of the AAA anticodon, there was significant misincorporation of a noncanonical amino acid into unwanted UUC Phe codons. To enhance codon selectivity, we explored degenerate leucine (Leu) codons instead of Phe degenerate codons. Combined use of the mutant yeast phenylalanyl-tRNA containing the CAA anticodon and the yPheRS_naph variant allowed incorporation of a phenylalanine analog, 2-naphthylalanine, into murine dihydrofolate reductase in response to multiple UUG Leu codons, but not to other Leu codon sites. Despite the moderate UUG codon occupancy by 2-naphthylalaine, these results successfully demonstrated that the concept of forced ambiguity of the genetic code can be achieved for the Leu codons, available for multiple-site-specific incorporation.     

Single sequence of a helix-loop peptide confers functional anticodon recognition on two tRNA synthetases.

The specific aminoacylation of RNA oligonucleotides whose sequences are based on the acceptor stems of tRNAs can be viewed as an operational RNA code for amino acids that may be related to the development of the genetic code. Many synthetases also have direct interactions with tRNA anticodon triplets and, in some cases, these interactions are thought to be essential for aminoacylation specificity. In these instances, an unresolved question is whether interactions with parts of the tRNA outside of the anticodon are sufficient for decoding genetic information. Escherichia coli isoleucyl- and methionyl-tRNA synthetases are closely related enzymes that interact with their respective anticodons. We used binary combinatorial mutagenesis of a 10 amino acid anticodon binding peptide in these two enzymes to identify composite sequences that would confer function to both enzymes despite their recognizing different anticodons. A single peptide was found that confers function to both enzymes in vivo and in vitro. Thus, even in enzymes where anticodon interactions are normally important for distinguishing one tRNA from another, these interactions can be 'neutralized' without losing specificity of amino-acylation. We suggest that acceptor helix interactions may play a role in providing the needed specificity.     

Expression of mutant alanine tRNAs increases spontaneous mutagenesis in Escherichia coli

The expression of mutA, an allele of the glycine tRNA gene glyV, can confer a novel mutator phenotype that correlates with its ability to promote Asp-->Gly mistranslation. Both activities are mediated by a single base change within the anticodon such that the mutant tRNA can decode aspartate codons (GAC/U) instead of the normal glycine codons (GCC/U). Here, we investigate whether specific Asp-->Gly mistranslation is required for the unexpected mutator phenotype. To address this question, we created and expressed 18 individual alleles of alaV, the gene encoding an alanine tRNA, in which the alanine anticodon was replaced with those specifying other amino acids such that the mutant (alaVX) tRNAs are expected to potentiate X-->Ala mistranslation, where X is one of the other amino acids. Almost all alaVX alleles proved to be mutators in an assay that measured the frequency of rifampicin-resistant mutants, with one allele (alaVGlu) being a stronger mutator than mutA. The alaVGlu mutator phenotype resembles that of mutA in mutational specificity (predominantly transversions), as well as SOS independence, but in a puzzling twist differs from mutA in that it does not require a functional recA gene. Our results suggest that general mistranslation (as opposed to Asp-->Gly alone) can induce a mutator phenotype. Furthermore, these findings predict that a large number of conditions that increase translational errors, such as genetic defects in the translational apparatus, as well as environmental and physiological stimuli (such as amino acid starvation or exposure to antibiotics) are likely to activate a mutator response. Thus, both genetic and epigenetic mechanisms can accelerate the acquisition of mutations.     

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.     

Unambiguous correspondence and complementarity of the chemical properties of amino acids and anticodons of the genetic code

The chemical language of genetic code is proposed. As a result of chemical language application for the analysis of the modern genetic code, the existence of an unambiguous correspondence between the chemical properties of amino acids and their coding triplets (codons and anticodons) is shown. This confirms the hypothesis of the code chemical determination. The complementarity between the chemical properties of amino acids and their anticodons (but not the codons) has been found also to exist. This observation supports the hypothesis of the genetic code determination by the direct recognition and also underlines the primary role of anticodon in the origin of genetic code in comparison with codons.     

Effects of Trypanosoma brucei tryptophanyl-tRNA synthetases silencing by RNA interference

The kinetoplast genetic code deviates from the universal code in that 90% of mitochondrial tryptophans are specified by UGA instead of UGG codons. A single nucleus-encoded tRNA Trp(CCA) is used by both nuclear and mitochondria genes, since all kinetoplast tRNAs are imported into the mitochondria from the cytoplasm. To allow decoding of the mitochondrial UGA codons as tryptophan, the tRNA Trp(CCA) anticodon is changed to UCA by an editing event. Two tryptophanyl tRNA synthetases (TrpRSs) have been identified in Trypanosoma brucei: TbTrpRS1 and TbTrpRS2 which localize to the cytoplasm and mitochondria respectively. We used inducible RNA interference (RNAi) to assess the role of TbTrpRSs. Our data validates previous observations of TrpRS as potential drug design targets and investigates the RNAi effect on the mitochondria of the parasite.     

Pyrrolysyl-tRNA synthetase: An ordinary enzyme but an outstanding genetic code expansion tool

The genetic incorporation of the 22nd proteinogenic amino acid, pyrrolysine (Pyl) at amber codon is achieved by the action of pyrrolysyl-tRNA synthetase (PylRS) together with its cognate tRNA^Pyl. Unlike most aminoacyl-tRNA synthetases, PylRS displays high substrate side chain promiscuity, low selectivity toward its substrate α-amine, and low selectivity toward the anticodon of tRNA^Pyl. These unique but ordinary features of PylRS as an aminoacyl-tRNA synthetase allow the Pyl incorporation machinery to be easily engineered for the genetic incorporation of more than 100 non-canonical amino acids (NCAAs) or α-hydroxy acids into proteins at amber codon and the reassignment of other codons such as ochre UAA, opal UGA, and four-base AGGA codons to code NCAAs.     

An integrated,structure- and energy-based view of the genetic code

The principles of mRNA decoding are conserved among all extant life forms. We present an integrative view of all the interaction networks between mRNA, tRNA and rRNA: the intrinsic stability of codon–anticodon duplex, the conformation of the anticodon hairpin, the presence of modified nucleotides, the occurrence of non-Watson–Crick pairs in the codon–anticodon helix and the interactions with bases of rRNA at the A-site decoding site. We derive a more information-rich, alternative representation of the genetic code, that is circular with an unsymmetrical distribution of codons leading to a clear segregation between GC-rich 4-codon boxes and AU-rich 2:2-codon and 3:1-codon boxes. All tRNA sequence variations can be visualized, within an internal structural and energy framework, for each organism, and each anticodon of the sense codons. The multiplicity and complexity of nucleotide modifications at positions 34 and 37 of the anticodon loop segregate meaningfully, and correlate well with the necessity to stabilize AU-rich codon–anticodon pairs and to avoid miscoding in split codon boxes. The evolution and expansion of the genetic code is viewed as being originally based on GC content with progressive introduction of A/U together with tRNA modifications. The representation we present should help the engineering of the genetic code to include non-natural amino acids.     

Evolution of the Genetic Code by Incorporation of Amino Acids that Improved or Changed Protein Function

Fifty years have passed since the genetic code was deciphered, but how the genetic code came into being has not been satisfactorily addressed. It is now widely accepted that the earliest genetic code did not encode all 20 amino acids found in the universal genetic code as some amino acids have complex biosynthetic pathways and likely were not available from the environment. Therefore, the genetic code evolved as pathways for synthesis of new amino acids became available. One hypothesis proposes that early in the evolution of the genetic code four amino acids—valine, alanine, aspartic acid, and glycine—were coded by GNC codons (N = any base) with the remaining codons being nonsense codons. The other sixteen amino acids were subsequently added to the genetic code by changing nonsense codons into sense codons for these amino acids. Improvement in protein function is presumed to be the driving force behind the evolution of the code, but how improved function was achieved by adding amino acids has not been examined. Based on an analysis of amino acid function in proteins, an evolutionary mechanism for expansion of the genetic code is described in which individual coded amino acids were replaced by new amino acids that used nonsense codons differing by one base change from the sense codons previously used. The improved or altered protein function afforded by the changes in amino acid function provided the selective advantage underlying the expansion of the genetic code. Analysis of amino acid properties and functions explains why amino acids are found in their respective positions in the genetic code.     

On the origin of the genetic code

It is argued that three chemical criteria determined the evolution of the genetic code: codon-anticodon pairing; codon-amino acid pairing; amino acid pairing. The first criterium determined the set of interactive nucleotides; the second, the set of nucleotides interactive with amino acids; the third, the set of mutually interactive amino acids. The code resulted from the intersection of these sets. This hypothesis explains the specificity and universality of the code as well as the “choice” of the standard amino acids and nucleotides from among those available in nature. The specific mechanism for codon-amino acid pairing assumed here is the “backwards” (Crick, 1967) Pelc-Welton (1966) models. Three types of evidence support “backwards” pairing: parallel genetic coding of amino acid pairs (Root-Bernstein, 1982); results of binding experiments by Saxinger and Ponnamperuma (1974); reinterpretation of Jungck's (1978) correlations between the properties of amino acids and their respective anticodon nucleotides. The inversion of the code to its present state occurred as a result of the evolution of tRNA molecules which supplanted parallel codon-amino acid interactions with antiparallel codon-anticodon ones. The paper concludes with suggestions for testing the hypothesis.     

Challenges of the genetic code for exploring sequence space in directed protein evolution

Directed protein evolution is the most versatile method for studying protein structure-function relationships, and for tailoring a protein's properties to the needs of industrial applications. In this review, we performed a statistical analysis on the genetic code to study the extent and consequence of the organization of the genetic code on amino acid substitution patterns generated in directed evolution experiments. In detail, we analyzed amino acid substitution patterns caused by (a) a single nucleotide (nt) exchange at each position of all 64 codons, and (b) two subsequent nt exchanges (first and second nt, first and third nt, second and third nt). Additionally, transitions and transversions mutations were compared at the level of amino acid substitution patterns. The latter analysis showed that single nucleotide substitution in a codon generates only 39.5% of the natural diversity on the protein level with 5.2-7 amino acid substitutions per codon. Transversions generate more complex amino acid substitution patterns (increased number and chemically more diverse amino acid substitutions) than transitions. Simultaneous nt exchanges at both first and second nt of a codon generates very diverse amino acid substitution patterns, achieving 83.2% of the natural diversity. The statistical analysis described in this review sets the objectives for novel random mutagenesis methods that address the consequences of the organization of the genetic code. Random mutagenesis methods that favor transversions or introduce consecutive nt exchanges can contribute in this regard.     

A more complex isoleucine aptamer with a cognate triplet

The simplest RNA that can meet a column affinity selection for isoleucine was previously defined using selection amplification with decreasing numbers of randomized nucleotides. This simplest UAUU motif was a small asymmetric internal loop. Conserved positions of the loop include isoleucine codon and anticodon triplets (Lozupone C., Changayil, S., Majerfeld, I., and Yarus, M. (2003) RNA (N. Y.) 9, 1315-1322). Using new primer sequences, we now select a somewhat more complex isoleucine binding RNA, requiring 4.7 more bits of information to describe. The newly selected structure is a terminal or hairpin loop of 20 nucleotides, 15 being invariant. An information profile shows that the new binding site contains five short functional loop regions joined by less significant single nucleotide positions. Among the important nucleotides is a conserved isoleucine anticodon, supporting the escaped triplet theory, which posits a stereochemical genetic code originating in RNA amino acid binding sites.     

RNA-ligand chemistry: a testable source for the genetic code

In the genetic code, triplet codons and amino acids can be shown to be related by chemical principles. Such chemical regularities could be created either during the code's origin or during later evolution. One such chemical principle can now be shown experimentally. Natural or particularly selected RNA binding sites for at least three disparate amino acids (arginine, isoleucine, and tyrosine) are enriched in codons for the cognate amino acid. Currently, in 517 total nucleotides, binding sites contain 2.4-fold more codon sequences than surrounding nucleotides. The aggregate probability of this enrichment is 10(-7) to 10(-8), had codons and binding site sequences been independent. Thus, at least some primordial coding assignments appear to have exploited triplets from amino acid binding sites as codons.     

Decoding of tandem quadruplets by adjacent tRNAs with eight-base anticodon loops

To expand the genetic code for specification of multiple non-natural amino acids, unique codons for these novel amino acids are needed. As part of a study of the potential of quadruplets as codons, the decoding of tandem UAGA quadruplets by an engineered tRNA^Leu with an eight-base anticodon loop, has been investigated. When GCC is the codon immediately 5′ of the first UAGA quadruplet, and release factor 1 is partially inactivated, the tandem UAGAs specify two leucines with an overall efficiency of at least 10%. The presence of a purine at anticodon loop position 32 of the tRNA decoding the codon 5′ to the first UAGA seems to influence translation of the following codon. Another finding is intraribosomal dissociation of anticodons from codons and their re-pairing to mRNA at overlapping or nearby codons. In one case where GCC is replaced by CGG, only a single Watson–Crick base pair can form upon re-pairing when decoding is resumed. This has implications for the mechanism of some cases of programmed frameshifting.     

Transfer RNA Identity Change in Anticodon Variants of E. coli tRNAPhe in Vivo

The anticodon sequence is a major recognition element for most aminoacyl-tRNA synthetases. We investigated the in vivo effects of changing the anticodon on the aminoacylation specificity in the example of E. coli tRNA^Phe. Constructing different anticodon mutants of E. coli tRNA^Phe by site-directed mutagenesis, we isolated 22 anticodon mutant tRNA^Phe; the anticodons corresponded to 16 amino acids and an opal stop codon. To examine whether the mutant tRNAs had changed their amino acid acceptor specificity in vivo, we tested the viability of E. coli strains containing these tRNA^Phe genes in a medium which permitted tRNA induction. Fourteen mutant tRNA genes did not affect host viability. However, eight mutant tRNA genes were toxic to the host and prevented growth, presumably because the anticodon mutants led to translational errors. Many mutant tRNAs which did not affect host viability were not aminoacylated in vivo. Three mutant tRNAs containing anticodon sequences corresponding to lysine (UUU), methionine (CAU) and threonine (UGU) were charged with the amino acid corresponding to their anticodon, but not with phenylalanine. These three tRNAs and tRNA^Phe are located in the same cluster in a sequence similarity dendrogram of total E. coli tRNAs. The results support the idea that such tRNAs arising from in vivo evolution are derived by anticodon change from the same ancestor tRNA.     

Challenges of the genetic code for exploring sequence space in directed protein evolution

Directed protein evolution is the most versatile method for studying protein structure–function relationships, and for tailoring a protein's properties to the needs of industrial applications. In this review, we performed a statistical analysis on the genetic code to study the extent and consequence of the organization of the genetic code on amino acid substitution patterns generated in directed evolution experiments. In detail, we analyzed amino acid substitution patterns caused by (a) a single nucleotide (nt) exchange at each position of all 64 codons, and (b) two subsequent nt exchanges (first and second nt, first and third nt, second and third nt). Additionally, transitions and transversions mutations were compared at the level of amino acid substitution patterns. The latter analysis showed that single nucleotide substitution in a codon generates only 39.5% of the natural diversity on the protein level with 5.2–7 amino acid substitutions per codon. Transversions generate more complex amino acid substitution patterns (increased number and chemically more diverse amino acid substitutions) than transitions. Simultaneous nt exchanges at both first and second nt of a codon generates very diverse amino acid substitution patterns, achieving 83.2% of the natural diversity. The statistical analysis described in this review sets the objectives for novel random mutagenesis methods that address the consequences of the organization of the genetic code. Random mutagenesis methods that favor transversions or introduce consecutive nt exchanges can contribute in this regard.     

On Nature’s Strategy for Assigning Genetic Code Multiplicity

Genetic code redundancy would yield, on the average, the assignment of three codons for each of the natural amino acids. The fact that this number is observed only for incorporating Ile and to stop RNA translation still waits for an overall explanation. Through a Structural Bioinformatics approach, the wealth of information stored in the Protein Data Bank has been used here to look for unambiguous clues to decipher the rationale of standard genetic code (SGC) in assigning from one to six different codons for amino acid translation. Leu and Arg, both protected from translational errors by six codons, offer the clearest clue by appearing as the most abundant amino acids in protein-protein and protein-nucleic acid interfaces. Other SGC hidden messages have been sought by analyzing, in a protein structure framework, the roles of over- and under-protected amino acids.