tRNA residues evolved to promote translational accuracy

The decoding properties of 22 structurally conservative base-pair and base-triple mutations in the anticodon hairpin and tertiary core of Escherichia coli tRNA^Ala_GGC were determined under single turnover conditions using E. coli ribosomes. While all of the mutations were able to efficiently decode the cognate GCC codon, many showed substantial misreading of near-cognate GUC or ACC codons. Although all the misreading mutations were present in the sequences of other E. coli tRNAs, they were never found among bacterial tRNA^Ala_GGC sequences. This suggests that the sequences of bacterial tRNA^Ala_GGC have evolved to avoid reading incorrect codons.     

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.     

Sequence similarity is more relevant than species specificity in probabilistic backtranslation

Background  

Backtranslation is the process of decoding a sequence of amino acids into the corresponding codons. All synthetic gene design systems include a backtranslation module. The degeneracy of the genetic code makes backtranslation potentially ambiguous since most amino acids are encoded by multiple codons. The common approach to overcome this difficulty is based on imitation of codon usage within the target species.     

The restriction of codon ambiguity on the basis of known variants

Summary The genetic code may be used to formulate the nucleotide sequence of a messenger RNA from the known amino acid sequence of a protein. Unfortunately, the degeneracy of the code means that there will be ambiguity in the nucleotide assignments in a third or more of the positions. A simple procedure is given that utilizes the information of known genetic variants to reduce that ambiguity. Problems associated with silent polymorphism are treated. The human alpha and beta hemoglobins are used to exemplify the technique. A total of 68 nucleotides in the two sequences are thereby made less ambiguous. One reduction leads to a nucleotide inconsistent with the result of the recently published beta hemoglobin sequence.     

Comparison of translation loads for standard and alternative genetic codes

Background  

The (almost) universality of the genetic code is one of the most intriguing properties of cellular life. Nevertheless, several variants of the standard genetic code have been observed, which differ in one or several of 64 codon assignments and occur mainly in mitochondrial genomes and in nuclear genomes of some bacterial and eukaryotic parasites. These variants are usually considered to be the result of non-adaptive evolution. It has been shown that the standard genetic code is preferential to randomly assembled codes for its ability to reduce the effects of errors in protein translation.     

Population Genetic Structure of 4,12:a:? Salmonella enterica Strains from Harbor Porpoises

According to pulsed-field gel electrophoresis (PFGE) typing, 4,12:a:− Salmonella enterica isolates from harbor porpoises are highly diverse. However, porpoise isolates belong to only two multilocus sequence types within the eBurst group 18 (eBG18) genetic cluster, which also includes S. enterica serovars Bispebjerg and Abortusequi. Isolates of other, serologically similar serovars belong to unrelated eBGs. These assignments to eBGs were supported by eBG-specific sequences of the flagellar gene fliC.     

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.     

Evolution of tRNA recognition systems and tRNA gene sequences

The aminoacylation of tRNAs by the aminoacyl-tRNA synthetases recapitulates the genetic code by dictating the association between amino acids and tRNA anticodons. The sequences of tRNAs were analyzed to investigate the nature of primordial recognition systems and to make inferences about the evolution of tRNA gene sequences and the evolution of the genetic code. Evidence is presented that primordial synthetases recognized acceptor stem nucleotides prior to the establishment of the three major phylogenetic lineages. However, acceptor stem sequences probably did not achieve a level of sequence diversity sufficient to faithfully specify the anticodon assignments of all 20 amino acids. This putative bottleneck in the evolution of the genetic code may have been alleviated by the advent of anticodon recognition. A phylogenetic analysis of tRNA gene sequences from the deep Archaea revealed groups that are united by sequence motifs which are located within a region of the tRNA that is involved in determining its tertiary structure. An association between the third anticodon nucleotide (N36) and these sequence motifs suggests that a tRNA-like structure existed close to the time that amino acid-anticodon assignments were being established. The sequence analysis also revealed that tRNA genes may evolve by anticodon mutations that recruit tRNAs from one isoaccepting group to another. Thus tRNA gene evolution may not always be monophyletic with respect to each isoaccepting group.Based on a presentation made at a workshop— Aminoacyl-tRNA Synthetases and the Evolution of the Genetic Code—held at Berkeley, CA, July 17–20, 1994 Correspondence to: M.E. Saks     

Sequential assignments in uniformly 13C- and 15N-labelled RNAs: The HC(N,P) and HC(N,P)-CCH-TOCSY experiments

Summary An approach for the simultaneous acquisition of HCN and HCP as well as HCN-CCH-TOCSY and HCP-CCH-TOCSY triple resonance data sets for ¹³C-/¹⁵N-labelled RNAs is presented. The new HCN-CCH-TOCSY scheme unambiguously links all sugar resonances to the base nitrogen. In addition, simultaneous acquisition of HCN-CCH-TOCSY and HCP-CCH-TOCSY data sets provides sequential and base-type information in a single experiment, thereby saving data acquisition time as well as providing complementary data sets that are useful in clarifying ambiguous assignments. Virtually complete sequence-specific phosphate-ribose ¹H, ³¹P, and base ¹⁵N1,9 assignments as well as partial ¹³C assignments could be obtained in a single experiment for a 0.5-mM sample of a 19-mer ribonucleotide.     

Molecular evidence for further overlooked species within the Gammarus fossarum complex (Crustacea: Amphipoda)

The freshwater amphipod Gammarus fossarum Koch, in Panzer, 1836 is a locally abundant keystone species mainly occuring in European headwaters but also in larger rivers. Genetic studies in the past 25 years have revealed three cryptic species within nominal G. fossarum (types A, B and C). Assignments of specimens to these types were based on allozyme and 16S markers. Today, a fragment of the cytochrome c oxidase subunit 1 (CO1) is primarily used as a genetic marker for species assignments (‘DNA Barcoding’), yet not a single CO1 sequence of G. fossarum is available in the Barcode of Life Database. We analysed new CO1 and 16S data for German, Hungarian and Croatian G. fossarum specimens and compared them with 16S and CO1 sequences of G. fossarum from GenBank. Thereby, we close the gap between traditional allozyme- and 16S-based species assignments and modern CO1 barcoding. Studying genetic variation in 55 specimens from 29 populations, we identified between 11 and 23 novel and genetically distinct clades using distance- and tree-based methods. Our results suggest that G. fossarum comprises several additional, yet unrecognised, species in particular from the Balkan region. Therefore, a taxonomic revision and biogeographic reconsideration of the G. fossarum complex is urgently needed.     

A Realistic Model Under Which the Genetic Code is Optimal

The genetic code has a high level of error robustness. Using values of hydrophobicity scales as a proxy for amino acid character, and the mean square measure as a function quantifying error robustness, a value can be obtained for a genetic code which reflects the error robustness of that code. By comparing this value with a distribution of values belonging to codes generated by random permutations of amino acid assignments, the level of error robustness of a genetic code can be quantified. We present a calculation in which the standard genetic code is shown to be optimal. We obtain this result by (1) using recently updated values of polar requirement as input; (2) fixing seven assignments (Ile, Trp, His, Phe, Tyr, Arg, and Leu) based on aptamer considerations; and (3) using known biosynthetic relations of the 20 amino acids. This last point is reflected in an approach of subdivision (restricting the random reallocation of assignments to amino acid subgroups, the set of 20 being divided in four such subgroups). The three approaches to explain robustness of the code (specific selection for robustness, amino acid–RNA interactions leading to assignments, or a slow growth process of assignment patterns) are reexamined in light of our findings. We offer a comprehensive hypothesis, stressing the importance of biosynthetic relations, with the code evolving from an early stage with just glycine and alanine, via intermediate stages, towards 64 codons carrying todays meaning.     

Population structure and cryptic genetic variation in the mango fruit fly,Ceratitis cosyra (Diptera,Tephritidae)

The fruit fly Ceratitis cosyra is an important agricultural pest negatively affecting the mango crop production throughout Africa and also feeding on a variety of other wild and cultivated hosts. The occurrence of deeply divergent haplotypes, as well as extensive morphological variability, previously suggested possible cryptic speciation within Ceratitis cosyra. Here we provide the first large-scale characterisation of the population structure of Ceratitis cosyra with the main objective of verifying cryptic genetic variation. A total of 348 specimens from 13 populations were genotyped at 16 polymorphic microsatellite loci. Hardy-Weinberg equilibrium (HWE) deviations were observed in 40.4% of locus-population combinations and suggested the occurrence of genetic substructuring within populations. Discriminant Analysis of Principal Components (DAPC) showed genetic divergence between the vast majority of vouchers from Burundi and Tanzania (plus a few outliers from other African countries) and all other specimens sampled. Individual Bayesian assignments confirmed the existence of two main genotypic groups also occurring in sympatry. These data provided further support to the hypothesis that Ceratitis cosyra might include cryptic species. However, additional integrative taxonomy, possibly combining morphological, ecological and physiological approaches, is required to provide the necessary experimental support to this model.     

The amino acid composition of proteins: a method of analysis.

Four quasiloglinear models are proposed for describing relationships between the amino acid composition of proteins and the structure of the genetic code. The models allow estimation of base frequencies in all three codon positions and can be used to investigate “interactions” between any two codon positions. The estimation procedure proposed by Ohta and Kimura (Genetics64 (1970), 387–395) is discussed and using two of the proposed quasiloglinear models an analysis of the amino acid composition of human cytochrome c is presented. The analysis suggests that of the six codons which code for leucine (CUU, CUC, CUA and CUG) do not occur in human cytochrome c.     

Natural reassignment of CUU and CUA sense codons to alanine in Ashbya mitochondria

The discovery of diverse codon reassignment events has demonstrated that the canonical genetic code is not universal. Studying coding reassignment at the molecular level is critical for understanding genetic code evolution, and provides clues to genetic code manipulation in synthetic biology. Here we report a novel reassignment event in the mitochondria of Ashbya (Eremothecium) gossypii, a filamentous-growing plant pathogen related to yeast (Saccharomycetaceae). Bioinformatics studies of conserved positions in mitochondrial DNA-encoded proteins suggest that CUU and CUA codons correspond to alanine in A. gossypii, instead of leucine in the standard code or threonine in yeast mitochondria. Reassignment of CUA to Ala was confirmed at the protein level by mass spectrometry. We further demonstrate that a predicted is transcribed and accurately processed in vivo, and is responsible for Ala reassignment. Enzymatic studies reveal that is efficiently recognized by A. gossypii mitochondrial alanyl-tRNA synthetase (AgAlaRS). AlaRS typically recognizes the G3:U70 base pair of tRNA^Ala; a G3A change in Ashbya abolishes its recognition by AgAlaRS. Conversely, an A3G mutation in Saccharomyces cerevisiae confers tRNA recognition by AgAlaRS. Our work highlights the dynamic feature of natural genetic codes in mitochondria, and the relative simplicity by which tRNA identity may be switched.     

Cover Picture: Biotechnology Journal 8/2017

Thaw what is frozen: it is generally accepted that the initial genetic code evolved from an ambiguous to a well‐defined (”frozen“) code with the repertoire of 20 (+2) canonical amino acids. If code is perfectly optimized by evolution, is it then possible to change it experimentally? If so, what are the barriers to overcome? Kubyshkin and Budisa have tried to answer this question by building a model that predicts ”entry points“ for invading the code with noncanonic amino acids. These experiments should lead to a chemical alienation of cells which represent an important step towards the creation of artificial life. The cover is prepared by Vladimir Kubyshkin and Nediljko Budisa authors of the article ”Synthetic alienation of microbial organisms by using genetic code engineering: Why and how?” ( https://doi.org/10.1002/biot.201600097 ).     

Evolution of the mitochondrial genetic code IV. AAA as an asparagine codon in some animal mitochondria

Summary Differences in assignments from those in the universal genetic code occur in codes of mitochondria. In this report, the published sequences of the mitochondrial genes for COI and ND1 in a platyhelminth (Fasciola hepatica) are examined and it is concluded that AAA may be a codon for asparagine instead of lysine, whereas AAG is the sole codon for lysine in this species.     

An algebraic hypothesis about the primeval genetic code architecture

A plausible architecture of an ancient genetic code is derived from an extended base triplet vector space over the Galois field of the extended base alphabet {D, A, C, G, U}, where symbol D represents one or more hypothetical bases with unspecific pairings. We hypothesized that the high degeneration of a primeval genetic code with five bases and the gradual origin and improvement of a primeval DNA repair system could make possible the transition from ancient to modern genetic codes. Our results suggest that the Watson-Crick base pairing G ≡ C and A = U and the non-specific base pairing of the hypothetical ancestral base D used to define the sum and product operations are enough features to determine the coding constraints of the primeval and the modern genetic code, as well as, the transition from the former to the latter. Geometrical and algebraic properties of this vector space reveal that the present codon assignment of the standard genetic code could be induced from a primeval codon assignment. Besides, the Fourier spectrum of the extended DNA genome sequences derived from the multiple sequence alignment suggests that the called period-3 property of the present coding DNA sequences could also exist in the ancient coding DNA sequences. The phylogenetic analyses achieved with metrics defined in the N-dimensional vector space (B³)^N of DNA sequences and with the new evolutionary model presented here also suggest that an ancient DNA coding sequence with five or more bases does not contradict the expected evolutionary history.     

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

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