Significance of genetic code module structure in gene expression and GC content enhancement in RNA sequences.

The existent algebraic models of the genetic code contribute to the understanding of the physio-chemical characteristics of the amino acids. However, the process of translating a gene into a phenotype is highly complex. Moreover, the intricacy of gene expression gets further multiplied due to the biases in the codon usage. This paper explores an algebraic structure called module on the set of codons as well as on that of RNA sequences. We study the potential implications of these structures on gene expression and the GC content of an RNA sequence. The base order {C,U,G,A} appears to possess greater biological significance than many of the orders previously studied. We have developed a novel algorithm to generate RNA sequences with high GC content, aiming to enhance the thermostability of biomolecules. The insights gained from this investigation may have applications in biomolecular modeling and docking, protein engineering, drug development, and related fields.

The tri-flow adaptiveness of codes in major evolutionary transitions.

Life codes increase in both number and variety with biological complexity. Although our knowledge of codes is constantly expanding, the evolutionary progression of organic, neural, and cultural codes in response to selection pressure remains poorly understood. Greater clarification of the selective mechanisms is achieved by investigating how major evolutionary transitions reduce spatiotemporal and energetic constraints on transmitting heritable code to offspring. Evolution toward less constrained flows is integral to enduring flow architecture everywhere, in both engineered and natural flow systems. Beginning approximately 4 billion years ago, the most basic level for transmitting genetic material to offspring was initiated by protocell division. Evidence from ribosomes suggests that protocells transmitted comma-free or circular codes, preceding the evolution of standard genetic code. This rudimentary information flow within protocells is likely to have first emerged within the geo-energetic and geospatial constraints of hydrothermal vents. A broad-gauged hypothesis is that major evolutionary transitions overcame such constraints with tri-flow adaptations. The interconnected triple flows incorporated energy-converting, spatiotemporal, and code-based informational dynamics. Such tri-flow adaptations stacked sequence splicing code on top of protein-DNA recognition code in eukaryotes, prefiguring the transition to sexual reproduction. Sex overcame the spatiotemporal-energetic constraints of binary fission with further code stacking. Examples are tubulin code and transcription initiation code in vertebrates. In a later evolutionary transition, language reduced metabolic-spatiotemporal constraints on inheritance by stacking phonetic, phonological, and orthographic codes. In organisms that reproduce sexually, each major evolutionary transition is shown to be a tri-flow adaptation that adds new levels of code-based informational exchange. Evolving biological complexity is also shown to increase the nongenetic transmissibility of code.

The time of appearance of the genetic code.

I support the hypothesis that the origin of the genetic code occurred simultaneously with the evolution of cellularity. That is to say, I favour the hypothesis that the origin of the genetic code is a very, very late event in the history of life on Earth. I corroborate this hypothesis with observations favouring the progenote's stage for the Last Universal Common Ancestor (LUCA), for the ancestor of bacteria and that of archaea. Indeed, these progenotic stages would imply that - at that time - the origin of the genetic code was still ongoing simply because this origin would fall within the very definition of progenote. Therefore, if the evolution of cellularity had truly been coeval with the origin of the genetic code - at least in its terminal part - then this would favour theories such as the coevolution theory of the origin of the genetic code because this theory would postulate that this origin must have occurred in extremely complex protocellular conditions and not concerning stereochemical or physicochemical interactions having to do with other stages of the origin of life. In this sense, the coevolution theory would be corroborated while the stereochemical and physicochemical theories would be damaged. Therefore, the origin of the genetic code would be linked to the origin of the cell and not to the origin of life as sometimes asserted. Therefore, I will discuss the late hypothesis of the origin of the genetic code in the context of the theories proposed to explain this origin and more generally of its implications for the early evolution of life.

Adding α,α-disubstituted and ß-linked monomers to the genetic code of an organism.

The genetic code of living cells has been reprogrammed to enable the site-specific incorporation of hundreds of non-canonical amino acids into proteins, and the encoded synthesis of non-canonical polymers and macrocyclic peptides and depsipeptides1-3. Current methods for engineering orthogonal aminoacyl-tRNA synthetases to acylate new monomers, as required for the expansion and reprogramming of the genetic code, rely on translational readouts and therefore require the monomers to be ribosomal substrates4-6. Orthogonal synthetases cannot be evolved to acylate orthogonal tRNAs with non-canonical monomers (ncMs) that are poor ribosomal substrates, and ribosomes cannot be evolved to polymerize ncMs that cannot be acylated onto orthogonal tRNAs-this co-dependence creates an evolutionary deadlock that has essentially restricted the scope of translation in living cells to α-L-amino acids and closely related hydroxy acids. Here we break this deadlock by developing tRNA display, which enables direct, rapid and scalable selection for orthogonal synthetases that selectively acylate their cognate orthogonal tRNAs with ncMs in Escherichia coli, independent of whether the ncMs are ribosomal substrates. Using tRNA display, we directly select orthogonal synthetases that specifically acylate their cognate orthogonal tRNA with eight non-canonical amino acids and eight ncMs, including several ß-amino acids, α,α-disubstituted-amino acids and ß-hydroxy acids. We build on these advances to demonstrate the genetically encoded, site-specific cellular incorporation of ß-amino acids and α,α-disubstituted amino acids into a protein, and thereby expand the chemical scope of the genetic code to new classes of monomers.

Dual stop codon suppression in mammalian cells with genomically integrated genetic code expansion machinery.

Stop codon suppression using dedicated tRNA/aminoacyl-tRNA synthetase (aaRS) pairs allows for genetically encoded, site-specific incorporation of non-canonical amino acids (ncAAs) as chemical handles for protein labeling and modification. Here, we demonstrate that piggyBac-mediated genomic integration of archaeal pyrrolysine tRNA (tRNAPyl)/pyrrolysyl-tRNA synthetase (PylRS) or bacterial tRNA/aaRS pairs, using a modular plasmid design with multi-copy tRNA arrays, allows for homogeneous and efficient genetically encoded ncAA incorporation in diverse mammalian cell lines. We assess opportunities and limitations of using ncAAs for fluorescent labeling applications in stable cell lines. We explore suppression of ochre and opal stop codons and finally incorporate two distinct ncAAs with mutually orthogonal click chemistries for site-specific, dual-fluorophore labeling of a cell surface receptor on live mammalian cells.

Evolution of the RNA world: From signals to codes.

The accumulated material in evolutionary biology, greatly enhanced by the achievements of modern synthetic biology, allows us to envision certain key hypothetical stages of prebiotic (chemical) evolution. This is often understood as the further evolution in the RNA World towards the RNA-protein World. It is a path towards the emergence of translation and the genetic code (I), signaling pathways with signaling molecules (II), and the appearance of RNA-based components of future gene regulatory networks (III). We believe that these evolutionary paths can be constructively viewed from the perspective of the concept of biological codes (Barbieri, 2003). Crucial evolutionary events in these directions would involve the emergence of RNA-based adaptors. Such adaptors connect two families of functionally and chemically distinct molecules into one functional entity. The emergence of primitive translation processes is undoubtedly the major milestone in the evolutionary path towards modern life. The key aspect here is the appearance of adaptors between amino acids and their cognate triplet codons. The initial steps are believed to involve the emergence of proto-transfer RNAs capable of self-aminoacylation. The second significant evolutionary breakthrough is the development of biochemical regulatory networks based on signaling molecules of the RNA World (ribonucleotides and their derivatives), as well as receptors and effectors (riboswitches) for these messengers. Some authors refer to this as the "lost language of the RNA World." The third evolutionary step is the emergence of signal sequences for ribozymes on the molecules of their RNA targets. This level of regulation in the RNA World is comparable to the gene regulatory networks of modern organisms. We believe that the signal sequences on target molecules have been rediscovered and developed by evolution into the gene regulatory networks of modern cells. In conclusion, the immense diversity of modern biological codes, in some of its key characteristics, can be traced back to the achievements of prebiotic evolution.

Karyotype as code of codes: An inheritance platform to shape the pattern and scale of evolution.

Organismal evolution displays complex dynamics in phase and scale which seem to trend towards increasing biocomplexity and diversity. For over a century, such amazing dynamics have been cleverly explained by the apparently straightforward mechanism of natural selection: all diversification, including speciation, results from the gradual accumulation of small beneficial or near-neutral alterations over long timescales. However, although this has been widely accepted, natural selection makes a crucial assumption that has not yet been validated. Specifically, the informational relationship between small microevolutionary alterations and large macroevolutionary changes in natural selection is unclear. To address the macroevolution-microevolution relationship, it is crucial to incorporate the concept of organic codes and particularly the "karyotype code" which defines macroevolutionary changes. This concept piece examines the karyotype from the perspective of two-phased evolution and four key components of information management. It offers insight into how the karyotype creates and preserves information that defines the scale and phase of macroevolution and, by extension, microevolution. We briefly describe the relationship between the karyotype code, the genetic code, and other organic codes in the context of generating evolutionary novelties in macroevolution and imposing constraints on them as biological routines in microevolution. Our analyses suggest that karyotype coding preserves many organic codes by providing system-level inheritance, and similar analyses are needed to classify and prioritize a large number of different organic codes based on the phases and scales of evolution. Finally, the importance of natural information self-creation is briefly discussed, leading to a call to integrate information and time into the relationship between matter and energy.

How biological codes break causal chains to enable autonomy for organisms.

Autonomy, meaning freedom from exogenous control, requires independence of both constitution and cybernetic regulation. Here, the necessity of biological codes to achieve both is explained, assuming that Aristotelian efficient cause is 'formal cause empowered by physical force'. Constitutive independence requires closure to efficient causation (in the Rosen sense); cybernetic independence requires transformation of cause-effect into signal-response relations at the organism boundary; the combination of both kinds of independence enables adaptation and evolution. Codes and cyphers translate information from one form of physical embodiment (domain) to another. Because information can only contribute as formal cause to efficient cause within the domain of its embodiment, translation can extend or restrict the range over which information is effective. Closure to efficient causation requires internalised information to be isolated from the cycle of efficient causes that it informs: e.g. Von Neumann self-replicator requires a (template) source of information that is causally isolated from the physical replication system. Life operationalises this isolation with the genetic code translating from the (isolated) domain of codons to that of protein interactions. Separately, cybernetic freedom is achieved at the cell boundary because transducers, which embody molecular coding, translate exogenous information into a domain where it no longer has the power of efficient cause. Information, not efficient cause, passes through the boundary to serve as stimulus for an internally generated response. Coding further extends freedom by enabling historically accumulated information to be selectively transformed into efficient cause under internal control, leaving it otherwise stored inactive. Code-based translation thus enables selective causal isolation, controlling the flow from cause to effect. Genetic code, cell-signalling codes and, in eukaryotes, the histone code, signal sequence based protein sorting and other code-dependent processes all regulate and separate causal chains. The existence of life can be seen as an expression of the power of molecular codes to selectively isolate and thereby organise causal relations among molecular interactions to form an organism.

Circular mixed sets.

In this article, we introduce the new mathematical concept of circular mixed sets of words over an arbitrary finite alphabet. These circular mixed sets may not be codes in the classical sense and hence allow a higher amount of information to be encoded. After describing their basic properties, we generalize a recent graph theoretical approach for circularity and apply it to distinguish codes from sets (i.e. non-codes). Moreover, several methods are given to construct circular mixed sets. Finally, this approach allows us to propose a new evolution model of the present genetic code that could have evolved from a dinucleotide world to a trinucleotide world via circular mixed sets of dinucleotides and trinucleotides.

Noncoding translation mitigation.

Translation is pervasive outside of canonical coding regions, occurring in long noncoding RNAs, canonical untranslated regions and introns1-4, especially in ageing4-6, neurodegeneration5,7 and cancer8-10. Notably, the majority of tumour-specific antigens are results of noncoding translation11-13. Although the resulting polypeptides are often nonfunctional, translation of noncoding regions is nonetheless necessary for the birth of new coding sequences14,15. The mechanisms underlying the surveillance of translation in diverse noncoding regions and how escaped polypeptides evolve new functions remain unclear10,16-19. Functional polypeptides derived from annotated noncoding sequences often localize to membranes20,21. Here we integrate massively parallel analyses of more than 10,000 human genomic sequences and millions of random sequences with genome-wide CRISPR screens, accompanied by in-depth genetic and biochemical characterizations. Our results show that the intrinsic nucleotide bias in the noncoding genome and in the genetic code frequently results in polypeptides with a hydrophobic C-terminal tail, which is captured by the ribosome-associated BAG6 membrane protein triage complex for either proteasomal degradation or membrane targeting. By contrast, canonical proteins have evolved to deplete C-terminal hydrophobic residues. Our results reveal a fail-safe mechanism for the surveillance of unwanted translation from diverse noncoding regions and suggest a possible biochemical route for the preferential membrane localization of newly evolved proteins.

Rare-event sampling analysis uncovers the fitness landscape of the genetic code.

The genetic code refers to a rule that maps 64 codons to 20 amino acids. Nearly all organisms, with few exceptions, share the same genetic code, the standard genetic code (SGC). While it remains unclear why this universal code has arisen and been maintained during evolution, it may have been preserved under selection pressure. Theoretical studies comparing the SGC and numerically created hypothetical random genetic codes have suggested that the SGC has been subject to strong selection pressure for being robust against translation errors. However, these prior studies have searched for random genetic codes in only a small subspace of the possible code space due to limitations in computation time. Thus, how the genetic code has evolved, and the characteristics of the genetic code fitness landscape, remain unclear. By applying multicanonical Monte Carlo, an efficient rare-event sampling method, we efficiently sampled random codes from a much broader random ensemble of genetic codes than in previous studies, estimating that only one out of every 1020 random codes is more robust than the SGC. This estimate is significantly smaller than the previous estimate, one in a million. We also characterized the fitness landscape of the genetic code that has four major fitness peaks, one of which includes the SGC. Furthermore, genetic algorithm analysis revealed that evolution under such a multi-peaked fitness landscape could be strongly biased toward a narrow peak, in an evolutionary path-dependent manner.

A swapped genetic code prevents viral infections and gene transfer.

Engineering the genetic code of an organism has been proposed to provide a firewall from natural ecosystems by preventing viral infections and gene transfer1-6. However, numerous viruses and mobile genetic elements encode parts of the translational apparatus7-9, potentially rendering a genetic-code-based firewall ineffective. Here we show that such mobile transfer RNAs (tRNAs) enable gene transfer and allow viral replication in Escherichia coli despite the genome-wide removal of 3 of the 64 codons and the previously essential cognate tRNA and release factor genes. We then establish a genetic firewall by discovering viral tRNAs that provide exceptionally efficient codon reassignment allowing us to develop cells bearing an amino acid-swapped genetic code that reassigns two of the six serine codons to leucine during translation. This amino acid-swapped genetic code renders cells resistant to viral infections by mistranslating viral proteomes and prevents the escape of synthetic genetic information by engineered reliance on serine codons to produce leucine-requiring proteins. As these cells may have a selective advantage over wild organisms due to virus resistance, we also repurpose a third codon to biocontain this virus-resistant host through dependence on an amino acid not found in nature10. Our results may provide the basis for a general strategy to make any organism safely resistant to all natural viruses and prevent genetic information flow into and out of genetically modified organisms.

The error minimization of the genetic code would have been determined by natural selection and not by a neutral evolution.

I discuss the mechanisms by which the error minimization observed in the genetic code would have been produced; that is, the ability of the genetic code to buffer, for example, the deleterious effects of translation errors. Here, I analyse whether the error minimization was produced by the intervention of natural selection or whether it is an emergent, that is, neutral property; in other words, whether it is a by-product of another mechanism that was structuring the genetic code. In particular, I criticize Massey's simulations (2008) - favouring the neutral hypothesis - which, containing elements of natural selection, would render his conclusions at least partly tautological. Furthermore, I criticize some of Koonin's (2017) interpretations regarding Massey's simulations. Finally, I criticize the opinion of Janzen et al. (2022) according to which their self-aminoacylating ribozyme system would have been capable of generating an error minimization of the genetic code as its emergent property. That is to say, I criticize, more generally, a neutral origin of error minimization. Indeed, any mechanism for structuring the genetic code would be capable of generating, in theory, such an emergent property. The problem is that to demonstrate this, it would be necessary to show that the level of optimization achieved by the genetic code would be that expected under the neutral hypothesis, the one that Janzen et al. (2022) instead they did not make. Therefore, their view is only a hypothesis and is very far from being corroborated by their results. Instead, in the literature there is a strong evidence that the level of optimization achieved by the genetic code is so high that it would imply, per se, an intervention of natural selection in the origin of error minimization of the genetic code. On the other hand, this level of optimization would be very far from what might have been produced by a neutral process.

Short tRNA anticodon stem and mutant eRF1 allow stop codon reassignment.

Cognate tRNAs deliver specific amino acids to translating ribosomes according to the standard genetic code, and three codons with no cognate tRNAs serve as stop codons. Some protists have reassigned all stop codons as sense codons, neglecting this fundamental principle1-4. Here we analyse the in-frame stop codons in 7,259 predicted protein-coding genes of a previously undescribed trypanosomatid, Blastocrithidia nonstop. We reveal that in this species in-frame stop codons are underrepresented in genes expressed at high levels and that UAA serves as the only termination codon. Whereas new tRNAsGlu fully cognate to UAG and UAA evolved to reassign these stop codons, the UGA reassignment followed a different path through shortening the anticodon stem of tRNATrpCCA from five to four base pairs (bp). The canonical 5-bp tRNATrp recognizes UGG as dictated by the genetic code, whereas its shortened 4-bp variant incorporates tryptophan also into in-frame UGA. Mimicking this evolutionary twist by engineering both variants from B. nonstop, Trypanosoma brucei and Saccharomyces cerevisiae and expressing them in the last two species, we recorded a significantly higher readthrough for all 4-bp variants. Furthermore, a gene encoding B. nonstop release factor 1 acquired a mutation that specifically restricts UGA recognition, robustly potentiating the UGA reassignment. Virtually the same strategy has been adopted by the ciliate Condylostoma magnum. Hence, we describe a previously unknown, universal mechanism that has been exploited in unrelated eukaryotes with reassigned stop codons.

High-yield recombinant bacterial expression of 13 C-, 15 N-labeled, serine-16 phosphorylated, murine amelogenin using a modified third generation genetic code expansion protocol.

Amelogenin constitutes ~90% of the enamel matrix in the secretory stage of amelogenesis, a still poorly understood process that results in the formation of the hardest and most mineralized tissue in vertebrates-enamel. Most biophysical research with amelogenin uses recombinant protein expressed in Escherichia coli. In addition to providing copious amounts of protein, recombinant expression allows 13 C- and 15 N-labeling for detailed structural studies using NMR spectroscopy. However, native amelogenin is phosphorylated at one position, Ser-16 in murine amelogenin, and there is mounting evidence that Ser-16 phosphorylation is important. Using a modified genetic code expansion protocol we have expressed and purified uniformly 13 C-, 15 N-labeled murine amelogenin (pS16M179) with ~95% of the protein being correctly phosphorylated. Homogeneous phosphorylation was achieved using commercially available, enriched, 13 C-, 15 N-labeled media, and protein expression was induced with isopropyl ß-D-1-thiogalactopyranoside at 310 K. Phosphoserine incorporation was verified from one-dimensional 31 P NMR spectra, comparison of 1 H-15 N HSQC spectra, Phos-tag SDS PAGE, and mass spectrometry. Phosphorus-31 NMR spectra for pS16M179 under conditions known to trigger amelogenin self-assembly into nanospheres confirm nanosphere models with buried N-termini. Lambda phosphatase treatment of these nanospheres results in the dephosphorylation of pS16M179, confirming that smaller oligomers and monomers with exposed N-termini are in equilibrium with nanospheres. Such 13 C-, 15 N-labeling of amelogenin with accurately encoded phosphoserine incorporation will accelerate biomineralization research to understand amelogenesis and stimulate the expanded use of genetic code expansion protocols to introduce phosphorylated amino acids into proteins.

Engineering enzyme activity using an expanded amino acid alphabet.

Enzyme design and engineering strategies are typically constrained by the limited size of nature's genetic alphabet, comprised of only 20 canonical amino acids. In recent years, site-selective incorporation of non-canonical amino acids (ncAAs) via an expanded genetic code has emerged as a powerful means of inserting new functional components into proteins, with hundreds of structurally diverse ncAAs now available. Here, we highlight how the emergence of an expanded repertoire of amino acids has opened new avenues in enzyme design and engineering. ncAAs have been used to probe complex biological mechanisms, augment enzyme function and, most ambitiously, embed new catalytic mechanisms into protein active sites that would be challenging to access within the constraints of nature's genetic code. We predict that the studies reviewed in this article, along with further advances in genetic code expansion technology, will establish ncAA incorporation as an increasingly important tool for biocatalysis in the coming years.

Recombinant and genetic code expanded collagen-like protein as a tailorable biomaterial.

Collagen occurs in nature with a dedicated triple helix structure and is the most preferred biomaterial in commercialized medical products. However, concerns on purity, disease transmission, and the reproducibility of animal derived collagen restrict its applications and warrants alternate recombinant sources. The expression of recombinant collagen in different prokaryotic and eukaryotic hosts has been reported with varying degrees of success, however, it is vital to elucidate the structural and biological characteristics of natural collagen. The recombinant production of biologically functional collagen is restricted by its high molecular weight and post-translational modification (PTM), especially the hydroxylation of proline to hydroxyproline. Hydroxyproline plays a key role in the structural stability and higher order self-assembly to form fibrillar matrices. Advancements in synthetic biology and recombinant technology are being explored for improving the yield and biomimicry of recombinant collagen. It emerges as reliable, sustainable source of collagen, promises tailorable properties and thereby custom-made protein biomaterials. Remarkably, the evolutionary existence of collagen-like proteins (CLPs) has been identified in single-cell organisms. Interestingly, CLPs exhibit remarkable ability to form stable triple helical structures similar to animal collagen and have gained increasing attention. Strategies to expand the genetic code of CLPs through the incorporation of unnatural amino acids promise the synthesis of highly tunable next-generation triple helical proteins required for the fabrication of smart biomaterials. The review outlines the importance of collagen, sources and diversification, and animal and recombinant collagen-based biomaterials and highlights the limitations of the existing collagen sources. The emphasis on genetic code expanded tailorable CLPs as the most sought alternate for the production of functional collagen and its advantages as translatable biomaterials has been highlighted.