Codon Assignments Research Articles

TRNA: The operational RNA code and protein folding

tRNA: The operational RNA code and protein folding Charles W. Carter, Jr., from the Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, relates molecular recognition used in genetic coding to structures of aminoacyl-tRNA synthetases and their cognate tRNAs. Genetic coding is simultaneously widely understood and little appreciated. Widely understood because Marshall Nierenberg worked out the codon assignments within a decade or so of the double helical structure of DNA by Watson and Crick (1). Little appreciated because a code, as far as we know, is something Nature had never produced – a symbolic mapping of the physical chemistry of the 20 amino acid side chains. The Morse code is a widely appreciated example. Like inheritance, the genetic code is a property of biology.

The Dimensionality of Genetic Information

This paper investigates the dimensionality of genetic information from the perspective of optimal representation. Recently it has been shown that optimal coding of information is in terms of the noninteger dimension of e, which is accompanied by the property of scale invariance. Since Nature is optimal, we should see this dimension reflected in the organization of the genetic code. With this as background, this paper investigates the problem of the logic behind the nature of the assignment of codons to amino acids, for they take different values that range from 1 to 6. It is shown that the non-uniformity of this assignment, which goes against mathematical coding theory that demands a near uniform assignment, is consistent with noninteger dimensions. The reason why the codon assignment for different amino acids varies is because uniformity is a requirement for optimality only in a standard vector space, and is not so in the noninteger dimensional space. It is noteworthy that there are 20 different covering regions in an e-dimensional information space, which is equal to the number of amino acids. The problem of the visualization of data that originates in an e-dimensional space but examined in a 3-dimensional vector space is also discussed. It is shown that the assignment of the codons to the amino acids is fractal-like that is well modeled by the Zipf distribution which is a power law. It is remarkable that the Zipf distribution that holds for the letter frequencies of words in a natural language also applies to the rank order of triplets in the code for amino acids.

The Genetic Code Assembles via Division and Fusion, Basic Cellular Events.

Standard Genetic Code (SGC) evolution is quantitatively modeled in up to 2000 independent coding 'environments'. Environments host multiple codes that may fuse or divide, with division yielding identical descendants. Code division may be selected-sophisticated gene products could be required for an orderly separation that preserves the coding. Several unforeseen results emerge: more rapid evolution requires unselective code division rather than its selective form. Combining selective and unselective code division, with/without code fusion, with/without independent environmental coding tables, and with/without wobble defines 25 = 32 possible pathways for SGC evolution. These 32 possible histories are compared, specifically, for evolutionary speed and code accuracy. Pathways differ greatly, for example, by ≈300-fold in time to evolve SGC-like codes. Eight of thirty-two pathways employing code division evolve quickly. Four of these eight that combine fusion and division also unite speed and accuracy. The two most precise, swiftest paths; thus the most likely routes to the SGC are similar, differing only in fusion with independent environmental codes. Code division instead of fusion with unrelated codes implies that exterior codes can be dispensable. Instead, a single ancestral code that divides and fuses can initiate fully encoded peptide biosynthesis. Division and fusion create a 'crescendo of competent coding', facilitating the search for the SGC and also assisting the advent of otherwise uniformly disfavored wobble coding. Code fusion can unite multiple codon assignment mechanisms. However, via code division and fusion, an SGC can emerge from a single primary origin via familiar cellular events.

Evolving a mitigation of the stress response pathway to change the basic chemistry of life

Despite billions of years of evolution, there have been only minor changes in the number and types of proteinogenic amino acids and the standard genetic code with codon assignments across the three domains of life. The rigidity of the genetic code sets it apart from other aspects of organismal evolution, giving rise to key questions about its origins and the constraints it places on innovation in translation. Through adaptive laboratory evolution (ALE) in Escherichia coli, we aimed to replace tryptophan (Trp) in the genetic code with an analogue L-β-(thieno[3,2-b]pyrrolyl)alanine ([3,2]Tpa). This required Escherichia coli to recruit thienopyrrole instead of indole and allowed reassignment of UGG codons. Crossing the stress response system emerged as a major obstacle for ancestral growth in the presence of [3,2]Tp and Trp limitation. During ALE, a pivotal innovation was the deactivation of the master regulon RpoS, which allowed growth solely in the presence of [3,2]Tp in minimal medium. Notably, knocking out the rpoS gene in the ancestral strain also facilitated growth on [3,2]Tp. Our findings suggest that regulatory constraints, not just a rigid translation mechanism, guard Life’s canonical amino acid repertoire. This knowledge will not only facilitate the design of more effective synthetic amino acid incorporation systems but may also shed light on a general biological mechanism trapping organismal configurations in a status quo.

Perseverance of protein homeostasis despite mistranslation of glycine codons with alanine.

By linking amino acids to their codon assignments, transfer RNAs (tRNAs) are essential for protein synthesis and translation fidelity. Some human tRNA variants cause amino acid mis-incorporation at a codon or set of codons. We recently found that a naturally occurring tRNASer variant decodes phenylalanine codons with serine and inhibits protein synthesis. Here, we hypothesized that human tRNA variants that misread glycine (Gly) codons with alanine (Ala) will also disrupt protein homeostasis. The A3G mutation occurs naturally in tRNAGly variants (tRNAGlyCCC, tRNAGlyGCC) and creates an alanyl-tRNA synthetase (AlaRS) identity element (G3 : U70). Because AlaRS does not recognize the anticodon, the human tRNAAlaAGC G35C (tRNAAlaACC) variant may function similarly to mis-incorporate Ala at Gly codons. The tRNAGly and tRNAAla variants had no effect on protein synthesis in mammalian cells under normal growth conditions; however, tRNAGlyGCC A3G depressed protein synthesis in the context of proteasome inhibition. Mass spectrometry confirmed Ala mistranslation at multiple Gly codons caused by the tRNAGlyGCC A3G and tRNAAlaAGC G35C mutants, and in some cases, we observed multiple mistranslation events in the same peptide. The data reveal mistranslation of Ala at Gly codons and defects in protein homeostasis generated by natural human tRNA variants that are tolerated under normal conditions. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

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.

Model of Genetic Code Structure Evolution under Various Types of Codon Reading

The standard genetic code (SGC) is a set of rules according to which 64 codons are assigned to 20 canonical amino acids and stop coding signal. As a consequence, the SGC is redundant because there is a greater number of codons than the number of encoded labels. This redundancy implies the existence of codons that encode the same genetic information. The size and organization of such synonymous codon blocks are important characteristics of the SGC structure whose evolution is still unclear. Therefore, we studied possible evolutionary mechanisms of the codon block structure. We conducted computer simulations assuming that coding systems at early stages of the SGC evolution were sets of ambiguous codon assignments with high entropy. We included three types of reading systems characterized by different inaccuracy and pattern of codon recognition. In contrast to the previous study, we allowed for evolution of the reading systems and their competition. The simulations performed under minimization of translational errors and reduction of coding ambiguity produced the coding system resistant to these errors. The reading system similar to that present in the SGC dominated the others very quickly. The survived system was also characterized by low entropy and possessed properties similar to that in the SGC. Our simulation show that the unambiguous SGC could emerged from a code with a lower level of ambiguity and the number of tRNAs increased during the evolution.

Average and Standard Deviation of the Error Function for Random Genetic Codes with Standard Stop Codons.

The origin of the genetic code has been attributed in part to an accidental assignment of codons to amino acids. Although several lines of evidence indicate the subsequent expansion and improvement of the genetic code, the hypothesis of Francis Crick concerning a frozen accident occurring at the early stage of genetic code evolution is still widely accepted. Considering Crick's hypothesis, mathematical descriptions of hypothetical scenarios involving a huge number of possible coexisting random genetic codes could be very important to explain the origin and evolution of a selected genetic code. This work aims to contribute in this regard, that is, it provides a theoretical framework in which statistical parameters of error functions are calculated. Given a genetic code and an amino acid property, the functional code robustness is estimated by means of a known error function. In this work, using analytical calculations, general expressions for the average and standard deviation of the error function distributions of completely random codes with standard stop codons were obtained. As a possible biological application of these results, any set of amino acids and any pure or mixed amino acid properties can be used in the calculations, such that, in case of having to select a set of amino acids to create a genetic code, possible advantages of natural selection of the genetic codes could be discussed.

Ribosome Fate during Decoding of UGA-Sec Codons.

Decoding of genetic information into polypeptides occurs during translation, generally following the codon assignment rules of the organism’s genetic code. However, recoding signals in certain mRNAs can overwrite the normal rules of translation. An exquisite example of this occurs during translation of selenoprotein mRNAs, wherein UGA codons are reassigned to encode for the 21st proteogenic amino acid, selenocysteine. In this review, we will examine what is known about the mechanisms of UGA recoding and discuss the fate of ribosomes that fail to incorporate selenocysteine.

The Combinatorial Fusion Cascade to Generate the Standard Genetic Code.

Combinatorial fusion cascade was proposed as a transition stage between prebiotic chemistry and early forms of life. The combinatorial fusion cascade consists of three stages: eight initial complimentary pairs of amino acids, four protocodes, and the standard genetic code. The initial complimentary pairs and the protocodes are divided into dominant and recessive entities. The transitions between these stages obey the same combinatorial fusion rules for all amino acids. The combinatorial fusion cascade mathematically describes the codon assignments in the standard genetic code. It explains the availability of amino acids with the even and odd numbers of codons, the appearance of stop codons, inclusion of novel canonical amino acids, exceptional high numbers of codons for amino acids arginine, leucine, and serine, and the temporal order of amino acid inclusion into the genetic code. The temporal order of amino acids within the cascade is congruent with the consensus temporal order previously derived from the similarities between the available hypotheses. The control over the combinatorial fusion cascades would open the road for a novel technology to develop artificial microorganisms.

Models of genetic code structure evolution with variable number of coded labels

It is assumed that at the early stage of cell evolution its translation machinery was characterized by high noise, i.e. ambiguous assignment of codons to amino acids in the genetic code, which initially encoded only few amino acids. Next, during its evolution new amino acids were added to this code. Taking into account this facts, we investigated theoretical models of genetic code’s structure, which evolved from a set of ambiguous codons assignments into a coding system with a low level of uncertainty. We considered three types of translational inaccuracies assuming a different number of fixed codon positions. We applied a modified version of evolutionary algorithm for finding the genetic codes that the most effectively reduced the initial uncertainty in the assignment of codons to encoded labels, i.e. amino acids and a stop translation signal. We examined codes with the number of labels from four to 22. Our results indicated that the quality of genetic code structure is strongly dependent on the number of encoded labels as well as the type of translational mechanism. The more strict assignments of codon to the labels was preferred by the codes encoding more number of labels. The results showed that a smaller degeneracy of codes evolved from a more tolerant coding with the stepwise addition of coded amino acids to the genetic code. The distribution of codon groups in the standard genetic code corresponds well to the translation model assuming two fixed codon positions, whereas the six-codon groups can be relics form previous stages of evolution when the code characterized by a greater uncertainty.

Fitting the standard genetic code into its triplet table

Minimally evolved codes are constructed here; these have randomly chosen standard genetic code (SGC) triplets, completed with completely random triplet assignments. Such "genetic codes" have not evolved, but retain SGC qualities. Retained qualities are basic, part of the underpinning of coding. For example, the sensitivity of coding to arbitrary assignments, which must be < ∼10%, is intrinsic. Such sensitivity comes from the elementary combinatorial properties of coding and constrains any SGC evolution hypothesis. Similarly, assignment of last-evolved functions is difficult because of late kinetic phenomena, likely common across codes. Census of minimally evolved code assignments shows that shape and size of wobble domains controls the code's fit into a coding table, strongly shifting accuracy of codon assignments. Access to the SGC therefore requires a plausible pathway to limited randomness, avoiding difficult completion while fitting a highly ordered, degenerate code into a preset three-dimensional space. Three-dimensional late Crick wobble in a genetic code assembled by lateral transfer between early partial codes satisfies these varied, simultaneous requirements. By allowing parallel evolution of SGC domains, this origin can yield shortened evolution to SGC-level order and allow the code to arise in smaller populations. It effectively yields full codes. Less obviously, it unifies previously studied chemical, biochemical, and wobble order in amino acid assignment, including a stereochemical minority of triplet-amino acid associations. Finally, fusion of intermediates into the final SGC is credible, mirroring broadly accepted later cellular evolution.

Optimal Evolution of the Standard Genetic Code

The Standard Genetic Code (SGC) exists in every known organism on Earth. SGC evolution via early unique codon assignment, then later wobble, yields coding resembling the near-universal code. Below, later wobble is shown to also create an optimal route to accurate codon assignment. Time of optimal codon assignment matches the previously defined mean time for ordered coding, exhibiting ≥ 90% of SGC order. Accurate evolution is also accessible, sufficiently frequent to appear in populations of 103 to 104 codes. SGC-like coding capacity, code order, and accurate assignments therefore arise together, in one attainable evolutionary intermediate. Examples, which plausibly resemble coding at evolutionary domain separation, are characterized.

C-skeleton in the genetic code from a basic series 5 → 0, guiding valences and other numeral features in the code

The search for regularities in the background for the genetic code and its codon assignments is here further developed, earlier shown to have many correlations with numeral series of integers 5 →0 with different exponents. The atomic mass analysis here counts on 20 + 4 double-coded amino acids, here including Ile AUA as such. A central finding here is that the C-skeleton seems to build on an hierarchical development of the mentioned basic series giving top numbers equal to those returning in side-chain divisions and on first three levels those of C-atoms in base-pair domains. It can very elementary explain the 3/2-division in the weight series. A few main results from earlier articles are shortly recapitulated, since it’s shown here that an x­3-series times 15 (x = integers 5 → 0) joins those earlier aspects and add new ones. It’s found also that atoms with valences 4 + 3 relative those with 2 + 1 make up a 3 to 1-division in both base-pair groups of codon domains, strengthening the earlier observation of valences as one important guiding principle in the relation between codons and domains of amino acids; valences of the atoms which in themselves make up a basic series 5 → 0 in the code when phosphorus P is included. Finally, fundamental factors in the code are gathered, where step 4 →3 seems reign at bottom of the code and number 7, exactly mean value of all atoms.

Combinatorial Fusion Rules to Describe Codon Assignment in the Standard Genetic Code

We propose combinatorial fusion rules that describe the codon assignment in the standard genetic code simply and uniformly for all canonical amino acids. These rules become obvious if the origin of the standard genetic code is considered as a result of a fusion of four protocodes: Two dominant AU and GC protocodes and two recessive AU and GC protocodes. The biochemical meaning of the fusion rules consists of retaining the complementarity between cognate codons of the small hydrophobic amino acids and large charged or polar amino acids within the protocodes. The proto tRNAs were assembled in form of two kissing hairpins with 9-base and 10-base loops in the case of dominant protocodes and two 9-base loops in the case of recessive protocodes. The fusion rules reveal the connection between the stop codons, the non-canonical amino acids, pyrrolysine and selenocysteine, and deviations in the translation of mitochondria. Using fusion rules, we predicted the existence of additional amino acids that are essential for the development of the standard genetic code. The validity of the proposed partition of the genetic code into dominant and recessive protocodes is considered referring to state-of-the-art hypotheses. The formation of two aminoacyl-tRNA synthetase classes is compatible with four-protocode partition.

Negative CG dinucleotide bias: An explanation based on feedback loops between Arginine codon assignments and theoretical minimal RNA rings

AbstractTheoretical minimal RNA rings are candidate primordial genes evolved for non‐redundant coding of the genetic code's 22 coding signals (one codon per biogenic amino acid, a start and a stop codon) over the shortest possible length: 29520 22‐nucleotide‐long RNA rings solve this min‐max constraint. Numerous RNA ring properties are reminiscent of natural genes. Here we present analyses showing that all RNA rings lack dinucleotide CG (a mutable, chemically instable dinucleotide coding for Arginine), bearing a resemblance to known CG‐depleted genomes. CG in “incomplete” RNA rings (not coding for all coding signals, with only 3–12 nucleotides) gradually decreases towards CG absence in complete, 22‐nucleotide‐long RNA rings. Presumably, feedback loops during RNA ring growth during evolution (when amino acid assignment fixed the genetic code) assigned Arg to codons lacking CG (AGR) to avoid CG. Hence, as a chemical property of base pairs, CG mutability restructured the genetic code, thereby establishing itself as genetically encoded biological information.

Codon assignment evolvability in theoretical minimal RNA rings.

Genetic code codon-amino acid assignments evolve for 15 (AAA, AGA, AGG, ATA, CGG, CTA, CTG. CTC, CTT, TAA, TAG, TCA, TCG, TGA and TTA (GNN codons notably absent)) among 64 codons (23.4%) across the 31 genetic codes (NCBI list completed with recently suggested green algal mitochondrial genetic codes). Their usage in 25 theoretical minimal RNA rings is examined. RNA rings are designed in silico to code once over the shortest length for all 22 coding signals (start and stop codons and each amino acid according to the standard genetic code). Though designed along coding constraints, RNA rings resemble ancestral tRNA loops, assigning to each RNA ring a putative anticodon, a cognate amino acid and an evolutionary genetic code integration rank for that cognate amino acid. Analyses here show 1. biases against/for evolvable codons in the two first vs last thirds of RNA ring coding sequences, 2. RNA rings with evolvable codons have recent cognates, and 3. evolvable codon and cytosine numbers in RNA ring compositions are positively correlated. Applying alternative genetic codes to RNA rings designed for nonredundant coding according to the standard genetic code reveals unsuspected properties of the standard genetic code and of RNA rings, notably on codon assignment evolvability and the special role of cytosine in relation to codon assignment evolvability and of the genetic code's coding structure.

Universal Codons with Enrichment from GC to AU Nucleotide Composition Reveal a Chronological Assignment from Early to Late Along with LUCA Formation.

The emergence of a primitive genetic code should be considered the most essential event during the origin of life. Almost a complete set of codons (as we know them) should have been established relatively early during the evolution of the last universal common ancestor (LUCA) from which all known organisms descended. Many hypotheses have been proposed to explain the driving forces and chronology of the evolution of the genetic code; however, none is commonly accepted. In the current paper, we explore the features of the genetic code that, in our view, reflect the mechanism and the chronological order of the origin of the genetic code. Our hypothesis postulates that the primordial RNA was mostly GC-rich, and this bias was reflected in the order of amino acid codon assignment. If we arrange the codons and their corresponding amino acids from GC-rich to AU-rich, we find that: 1. The amino acids encoded by GC-rich codons (Ala, Gly, Arg, and Pro) are those that contribute the most to the interactions with RNA (if incorporated into short peptides). 2. This order correlates with the addition of novel functions necessary for the evolution from simple to longer folded peptides. 3. The overlay of aminoacyl-tRNA synthetases (aaRS) to the amino acid order produces a distinctive zonal distribution for class I and class II suggesting an interdependent origin. These correlations could be explained by the active role of the bridge peptide (BP), which we proposed earlier in the evolution of the genetic code.

The influence of different types of translational inaccuracies on the genetic code structure

BackgroundThe standard genetic code is a recipe for assigning unambiguously 21 labels, i.e. amino acids and stop translation signal, to 64 codons. However, at early stages of the translational machinery development, the codons did not have to be read unambiguously and the early genetic codes could have contained some ambiguous assignments of codons to amino acids. Therefore, the goal of this work was to obtain the genetic code structures which could have evolved assuming different types of inaccuracy of the translational machinery starting from unambiguous assignments of codons to amino acids.ResultsWe developed a theoretical model assuming that the level of uncertainty of codon assignments can gradually decrease during the simulations. Since it is postulated that the standard code has evolved to be robust against point mutations and mistranslations, we developed three simulation scenarios assuming that such errors can influence one, two or three codon positions. The simulated codes were selected using the evolutionary algorithm methodology to decrease coding ambiguity and increase their robustness against mistranslation.ConclusionsThe results indicate that the typical codon block structure of the genetic code could have evolved to decrease the ambiguity of amino acid to codon assignments and to increase the fidelity of reading the genetic information. However, the robustness to errors was not the decisive factor that influenced the genetic code evolution because it is possible to find theoretical codes that minimize the reading errors better than the standard genetic code.

Many alternative and theoretical genetic codes are more robust to amino acid replacements than the standard genetic code

We evaluated the differences between the standard genetic code (SGC) and its known alternative variants in terms of the consequences of amino acids replacements. Furthermore, the properties of all the possible theoretical genetic codes, which differ from the SGC by one, two or three changes in codon assignments were also tested. Although the SGC is closer to the best theoretical codes than to the worst ones due to the minimization of amino acid replacements, from 10% to 27% of the all possible theoretical codes minimize the effect of these replacements better than the SGC. Interestingly, many types of codon reassignments observed in the alternative codes are also responsible for the substantial robustness to amino acid replacements. As many as 18 out of 21 alternatives perform better than the SGC under the assumed optimization criteria. These findings suggest that not all reassignments in the alternative codes are neutral and some of them could be selected to reduce harmful effects of mutations or translation of protein-coding sequences. The results also imply that the standard genetic code can be improved in this respect by a quite small number of changes, which are in fact realized in its variants. It would mean that the tendency to minimize mutational errors was not the main force that drove the evolution of the SGC.