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Since the establishment of site-specific mutagenesis of single amino acids to interrogate protein function in the 1970s, biochemists have sought to tailor protein structure in the native cell environment. Fine-tuning the chemical properties of proteins is an indispensable way to address fundamental mechanistic questions. Unnatural amino acids (UAAs) offer the possibility to expand beyond the 20 naturally occurring amino acids in most species and install new and useful chemical functions. Here, we review the literature about advances in UAA incorporation technology from chemoenzymatic aminoacylation of modified tRNAs to in vitro translation systems to genetic encoding of UAAs in the native cell environment and whole organisms. We discuss innovative applications of the UAA technology to challenges in bioengineering and medicine.

Biological macromolecules encode information: some of it to endow the molecule with structural flexibility, some of it to enable molecular actions as a catalyst or a substrate, but a residual part can be used to communicate with other macromolecules. Thus, macromolecules do not need to possess information only to survive in an environment, but also to strategically interact with others by sending signals to a receiving macromolecule that can properly interpret the signal and act suitably. These sender-receiver signalling games are sustained by the information asymmetry that exists among the macromolecules. In both biochemistry and molecular evolution, the important role of information asymmetry remains largely unaddressed. Here, we provide a new unifying perspective on the impact of information symmetry between macromolecules on molecular evolutionary processes, while focusing on molecular deception. Biomolecular games arise from the ability of biological macromolecules to exert precise recognition, and their role as units of selection, meaning that they are subject to competition and cooperation with other macromolecules. Thus, signalling game theory can be used to better understand fundamental features of living systems such as molecular recognition, molecular mimicry, selfish elements and 'junk' DNA. We show how deceptive behaviour at the molecular level indicates a conflict of interest, and so provides evidence of genetic conflict. This model proposes that molecular deception is diagnostic of selfish behaviour, helping to explain the evasive behaviour of transposable elements in 'junk' DNA, for example. Additionally, in this broad review, a range of major evolutionary transitions are shown to be associated with the establishment of signalling conventions, many of which are susceptible to molecular deception. These perspectives allow us to assign rudimentary behaviour to macromolecules, and show how participation in signalling games differentiates biochemistry from abiotic chemistry., (© 2018 The Author(s).)

The present study is just an overview of the opening of the geochemical stage for the appearance of life. But that opening would not have been sufficient for the intellectual discovery of the origin of life! The excellent works and many commendable efforts that advance this explanation have not shown the fundamental elements that participate in the theoretical frame of biological evolution. The latter imply the existence of evolutionary transitions and the production of new levels of organization. In this brief analysis we do not intend to introduce the audience to the philosophy of biology. But we do expect to provide a modest overview, in which the geochemical chemolithoautotrophic opening of the stage should be seen, at most, as the initial metabolism that enabled organic compounds to follow the road where a chemical fluid machinery was thus able to undertake the more "sublime" course of organic biological evolution. We think that Tibor Gánti's chemoton is the most significant contribution to theoretical biology, and the only course now available to comprehend the unit of evolution problem without the structuralist and functionalist conflict prevalent in theoretical biology. In our opinion Gánti's chemoton theory travels to the "locus" where evolutionary theory dares to extend itself to entities at many levels of structural organization, beyond the gene or the group above. Therefore, in this and subsequent papers on the prebiotic conditions for the eventual appearance of the genetic code, we explore the formation and the presence of metal sulfide minerals, from the assembly of metal sulfide clusters through the precipitation of nanocrystals and the further reactions resulting in bulk metal sulfide phases. We endeavor to characterize pristine reactions and the modern surfaces, utilizing traditional surface science techniques and computational methods. Moreover, mechanistic details of the overall oxidation of metal sulfide minerals are set forth. We hope that this paper will lead our audience to accept that in a chemically oscillating system the chemoton is a model fluid state automaton capable of growth and self-reproduction. This is not simply a matter of transmitting a pattern, as in inorganic crystals; such self-reproduction must be more complex than crystal growth. Indeed that is what Gánti's theoretical and abstract model offers to us all: we finally have a philosophy of evolutionary units in theoretical biology.

This lecture consists of a short appraisal of some of the main features that have characterized the growth of biochemistry during the course of the 20th century. It dwells on the early impacts of vitalism, the emergence and elucidation of the vitamins, the discovery of coenzymes, the concept of active centres of enzymes, the development of experimental techniques (including the use of isotopes), the genetic code, and on the development of molecular biology and closely allied fields of investigation. It concludes with a consideration of the influence of the study of membranes and of neurochemistry on current biochemical thought.

Two missense mutations, trpA58 and trpA78, and one nonsense mutation-trp-ochre, were used to determine the types of base-pair substitution caused by ultra, violet irradiation and methyl methanesulfonate (MMS) in Escherichia coli. UV irradiation of the wild-type bacteria led to the formation of revertants mainly arising as a result of GC yields AT transitions (suppressor revertants of the trpA58 mutant). True revertants of the trp- mutant (arising via transitions of AT pairs) and 5-methyl tryptophan-sensitive (MT-s) Trp+ of the trpA78 mutant (arising via unidentified transversions) occurred at a lower frequency. The polAI mutation did not change the frequency of the UV-induced transitions GC yields AT or that of the substitutions of the AT pairs. The uvrE502 mutation significantly increased the frequency of the UV-induced revertants arising via the transition GC yields AT. Treatment of the wild-type bacteria with MMS resulted in the formation of revertants mainly due to the GC yields AT substitution, and with a lower frequency to the AT yields GC transitions. MMS also induced, with a low frequency, some transversions. The frequency of the MMS-induced GC yields AT transitions was enhanced in the uvrE502 mutant. On the other hand, the uvrE502 mutation eliminated or significantly lowered MMS-induced revertants arising as a result of AT yields GC transitions or transversions.

Sulfur fluoride exchange (SuFEx) and phosphorus fluoride exchange (PFEx) click chemistries are advancing research across multiple disciplines. By genetically incorporating latent bioreactive unnatural amino acids (Uaas), these chemistries have been integrated into proteins, enabling precise covalent linkages with biological macromolecules and paving the way for new applications. However, their suboptimal reaction rates in proteins limit effectiveness, and traditional catalytic methods for small molecules are often incompatible with biological systems or in vivo applications. We demonstrated that introducing an arginine adjacent to the latent bioreactive Uaa significantly boosts SuFEx and PFEx reaction rates between proteins. This method is effective across various Uaas, target residues, and protein environments. Notably, it also enables efficient SuFEx reactions in acidic conditions, common in certain cellular compartments and tumor microenvironments, which typically hinder SuFEx reactions. Furthermore, we developed the first covalent cell engager that substantially enhances natural killer cell activation through improved covalent interaction facilitated by arginine. These findings provide mechanistic insights and offer a biocompatible strategy to harness these robust chemistries for advancing biological research and developing new biotherapeutics. [ABSTRACT FROM AUTHOR]

Transfer RNAs have been extensively explored as the molecules that translate the genetic code into proteins. At this interface of genetics and biochemistry, tRNAs direct the efficiency of every major step of translation by interacting with a multitude of binding partners. However, due to the variability of tRNA sequences and the abundance of diverse post-transcriptional modifications, a guidebook linking tRNA sequences to specific translational outcomes has yet to be elucidated. Here, we review substantial efforts that have collectively uncovered tRNA engineering principles that can be used as a guide for the tuning of translation fidelity. These principles have allowed for the development of basic research, expansion of the genetic code with non-canonical amino acids, and tRNA therapeutics. [ABSTRACT FROM AUTHOR]

Would another origin of life resemble Earth's biochemical use of amino acids? Here, we review current knowledge at three levels: (1) Could other classes of chemical structure serve as building blocks for biopolymer structure and catalysis? Amino acids now seem both readily available to, and a plausible chemical attractor for, life as we do not know it. Amino acids thus remain important and tractable targets for astrobiological research. (2) If amino acids are used, would we expect the same L-alpha-structural subclass used by life? Despite numerous ideas, it is not clear why life favors L-enantiomers. It seems clearer, however, why life on Earth uses the shortest possible (alpha-) amino acid backbone, and why each carries only one side chain. However, assertions that other backbones are physicochemically impossible have relaxed into arguments that they are disadvantageous. (3) Would we expect a similar set of side chains to those within the genetic code? Many plausible alternatives exist. Furthermore, evidence exists for both evolutionary advantage and physicochemical constraint as explanatory factors for those encoded by life. Overall, as focus shifts from amino acids as a chemical class to specific side chains used by post-LUCA biology, the probable role of physicochemical constraint diminishes relative to that of biological evolution. Exciting opportunities now present themselves for laboratory work and computing to explore how changing the amino acid alphabet alters the universe of protein folds. Near-term milestones include: (a) expanding evidence about amino acids as attractors within chemical evolution; (b) extending characterization of other backbones relative to biological proteins; and (c) merging computing and laboratory explorations of structures and functions unlocked by xeno peptides. [ABSTRACT FROM AUTHOR]

The Montessori method was an educational model devised at the beginning of the 20th century by the Italian pedagogue, physician, psychologist, and psychiatrist María Montessori (1870-1952). She observed that her students activated their intelligence and developed their personality by carrying out manual activities. In recent years, one of the activities that she used, gamification, is currently booming in teaching. Games based in words or letters are often used in language learning, either in presential lessons or in mobile applications. In our teaching innovation group (GINDOC-UB/180), we proposed to adapt those word games using codes so that students could achieve a self-learning on the nomenclature of biomolecules, thus improving their knowledge in metabolism. These code-based games were proposed to Biochemistry students at the Chemistry degree of the Universitat de Barcelona. Games were posted on the Virtual Campus using Moodle. A code is part of a communicative system, and it is defined as a set of elements that allows a message to be decoded. In biochemistry, the best-known code is the genetic code that translates 3 nucleotides to an amino acid. The code allows the students to see that it is a degenerated code, and it was the first code-based game used. Nevertheless, only 20 amino acids are involved, and some letters are missing. Thus, we also used other codes such as numeric codes, Morse code, Braille system code, or codes from other alphabets (Japanese katakana or hiragana, Cyrillic alphabet, Arabic alphabet, or runic alphabet). In this work some examples of the games that can be proposed to the students are shown. Proposed games were highly valued by students and allowed a self-learning on the proposed biomolecules. Games based on the genetic code were easier and more enjoyable for the students to solve, and they provided them with additional biochemical knowledge of protein translation. [ABSTRACT FROM AUTHOR]

This review presents the life stories of M. Nirenberg, H. Khorana, and R. Holley, winners of the 1968 Nobel Prize in Physiology or Medicine, the history of the discoveries made by these scientists, and the methodological approaches used in their works. Owing to the M. Nirenberg and H. Khorana research, the nucleotide compositions of all mRNA triplet codons were decoded. H. Khorana was the first scientist to experimentally prove the direct link between the nucleotide sequence of DNA and the amino acid sequence of the synthesized protein and to obtain a synthetic gene. R. Holley was the first to completely decode the sequence of transport RNA, determine its secondary structure and role in protein synthesis on the ribosome. The Nobel Prize awarded to the scientists was a recognition of their contribution in understanding the mechanisms of coding and reading genetic information and marked a breakthrough moment in the development of molecular biology.

The diversity of the genetic code systems used by microbes on earth is yet to be elucidated. It is known that certain methanogenic archaea employ an alternative system for cysteine (Cys) biosynthesis and encoding; tRNACys is first acylated with phosphoserine (Sep) by O-phosphoseryl-tRNA synthetase (SepRS) and then converted to Cys-tRNACys by Sep-tRNA:Cys-tRNA synthase (SepCysS). In this study, we searched all genomic and metagenomic protein sequence data in the Integrated Microbial Genomes (IMG) system and at the NCBI to reveal new clades of SepRS and SepCysS proteins belonging to diverse archaea in the four major groups (DPANN, Euryarchaeota, TACK, and Asgard) and two groups of bacteria ("Candidatus Parcubacteria" and Chloroflexi). Bacterial SepRS and SepCysS charged bacterial tRNACys species with cysteine in vitro Homologs of SepCysE, a scaffold protein facilitating SepRS⋅SepCysS complex assembly in Euryarchaeota class I methanogens, are found in a few groups of TACK and Asgard archaea, whereas the C-terminally truncated homologs exist fused or genetically coupled with diverse SepCysS species. Investigation of the selenocysteine (Sec)- and pyrrolysine (Pyl)-utilizing traits in SepRS-utilizing archaea and bacteria revealed that the archaea carrying full-length SepCysE employ Sec and that SepRS is often found in Pyl-utilizing archaea and Chloroflexi bacteria. We discuss possible contributions of the SepRS-SepCysS system for sulfur assimilation, methanogenesis, and other metabolic processes requiring large amounts of iron-sulfur enzymes or Pyl-containing enzymes.IMPORTANCE Comprehensive analyses of all genomic and metagenomic protein sequence data in public databases revealed the distribution and evolution of an alternative cysteine-encoding system in diverse archaea and bacteria. The finding that the SepRS-SepCysS-SepCysE- and the selenocysteine-encoding systems are shared by the Euryarchaeota class I methanogens, the Crenarchaeota AK8/W8A-19 group, and an Asgard archaeon suggests that ancient archaea may have used both systems. In contrast, bacteria may have obtained the SepRS-SepCysS system from archaea. The SepRS-SepCysS system sometimes coexists with a pyrrolysine-encoding system in both archaea and bacteria. Our results provide additional bioinformatic evidence for the contribution of the SepRS-SepCysS system for sulfur assimilation and diverse metabolisms which require vast amounts of iron-sulfur enzymes and proteins. Among these biological activities, methanogenesis, methylamine metabolism, and organohalide respiration may have local and global effects on earth. Taken together, uncultured bacteria and archaea provide an expanded record of the evolution of the genetic code.

Aminoacyl-tRNA synthetases use a variety of mechanisms to ensure fidelity of the genetic code and ultimately select the correct amino acids to be used in protein synthesis. The physiological necessity of these quality control mechanisms in different environments remains unclear, as the cost vs benefit of accurate protein synthesis is difficult to predict. We show that in Escherichia coli, a non-coded amino acid produced through oxidative damage is a significant threat to the accuracy of protein synthesis and must be cleared by phenylalanine-tRNA synthetase in order to prevent cellular toxicity caused by mis-synthesized proteins. These findings demonstrate how stress can lead to the accumulation of non-canonical amino acids that must be excluded from the proteome in order to maintain cellular viability.

Aminoacyl-tRNA synthetases use a variety of mechanisms to ensure fidelity of the genetic code and ultimately select the correct amino acids to be used in protein synthesis. The physiological necessity of these quality control mechanisms in different environments remains unclear, as the cost vs benefit of accurate protein synthesis is difficult to predict. We show that in Escherichia coli, a non-coded amino acid produced through oxidative damage is a significant threat to the accuracy of protein synthesis and must be cleared by phenylalanine-tRNA synthetase in order to prevent cellular toxicity caused by mis-synthesized proteins. These findings demonstrate how stress can lead to the accumulation of non-canonical amino acids that must be excluded from the proteome in order to maintain cellular viability.

• To change the genetic code, we need to understand how it was originally set. • The amino acid repertoire of the genetic code consists mainly of derivatives of alanine. • The Alanine World is a basis for α-helix dominated protein biochemistry of life on Earth. • Glycine, proline and pyrrolysine with derivatives have a potential to provide an alternative to the Alanine World. • They give rise to scaffolds that will be useful for the construction of synthetic life. Can we make life with a different genetic amino acid repertoire? Can we expect organisms which would keep newly given genetic code associations permanently? To address these questions, we would like to analyze the existent genetic code amino acid repertoire as formed from derivatives of alanine. Derivation from alanine leads to the α-helix based biological world, the Alanine World , whereas variations in the side-chains enable tertiary folding and subsequent chemical versatility of the proteome. Proline, glycine and pyrrolysine are the rudiments in the current genetic code, indicating that the original set could be different. Furthermore, from the perspective of peptide chemistry, it shall be possible to recruit these alternative scaffolds for the construction of synthetic or alternative life. This would allow for a completely new biological world, potentially as functional and versatile as the existing one. Pursuing these options offers a strategy for a complete re-design or even de-novo creation of living organisms based on entirely different chemical make-up, with completely new set of solutions for both near and distant future biotechnologies. [ABSTRACT FROM AUTHOR]

HIV-1 replicates via a low-fidelity polymerase with a high mutation rate; strong conservation of individual nucleotides is highly indicative of the presence of critical structural or functional properties. Identifying such conservation can reveal novel insights into viral behaviour. We analysed 3651 publicly available sequences for the presence of nucleic acid conservation beyond that required by amino acid constraints, using a novel scale-free method that identifies regions of outlying score together with a codon scoring algorithm. Sequences with outlying score were further analysed using an algorithm for producing local RNA folds whilst accounting for alignment properties. 11 different conserved regions were identified, some corresponding to well-known cis-acting functions of the HIV-1 genome but also others whose conservation has not previously been noted. We identify rational causes for many of these, including cis functions, possible additional reading frame usage, a plausible mechanism by which the central polypurine tract primes second-strand DNA synthesis and a conformational stabilising function of a region at the 5′ end of env. [ABSTRACT FROM AUTHOR]