Structural biology in translation system of genetic code

Nearly 50 years ago, Francis Crick propounded the frozen accident scenario for the evolution of the genetic code along with the hypothesis that the early translation system consisted primarily of RNA. Under the frozen accident perspective, the code is universal among modern life forms because any change in codon assignment would be highly deleterious. The frozen accident can be considered the default theory of code evolution because it does not imply any specific interactions between amino acids and the cognate codons or anticodons, or any particular properties of the code. The subsequent 49 years of code studies have elucidated notable features of the standard code, such as high robustness to errors, but failed to develop a compelling explanation for codon assignments. In particular, stereochemical affinity between amino acids and the cognate codons or anticodons does not seem to account for the origin and evolution of the code. Here I expand Crick’s hypothesis on RNA-only translation system by presenting evidence that this early translation already attained high fidelity that allowed protein evolution. I outline an experimentally testable scenario for the evolution of the code that combines a distinct version of the stereochemical hypothesis, in which amino acids are recognized via unique sites in the tertiary structure of proto-tRNAs, rather than by anticodons, expansion of the code via proto-tRNA duplication and the frozen accident.

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Broadening the toolkit for quantitatively evaluating noncanonical amino acid incorporation in yeast

10.1101/2021.08.02.454837 ◽

2021 ◽

Author(s):

Jessica T. Stieglitz ◽

Kelly A. Potts ◽

James A. Van Deventer

Keyword(s):

Genetic Code ◽

Fluorescent Protein ◽

Yeast Cells ◽

Translation System ◽

Critical Fields ◽

Noncanonical Amino Acids ◽

Eukaryotic Translation ◽

Genomic Changes ◽

Potential Applications ◽

Genetic Code Expansion

Genetic code expansion is a powerful approach for advancing critical fields such as biological therapeutic discovery. However, the machinery for genetically encoding noncanonical amino acids (ncAAs) is only available in limited plasmid formats, constraining potential applications. In extreme cases, the introduction of two separate plasmids, one containing an orthogonal translation system (OTS) to facilitate ncAA incorporation and a second for expressing a ncAA-containing protein of interest, is not possible due to lack of convenient selection markers. One strategy to circumvent this challenge is to express the OTS and protein of interest from a single vector. For what we believe is the first time in yeast, we describe here several sets of single plasmid systems (SPSs) for performing genetic code manipulation and compare the ncAA incorporation capabilities of these plasmids against the capabilities of previously described dual plasmid systems (DPSs). For both dual fluorescent protein reporters and yeast display reporters tested with multiple OTSs and ncAAs, measured ncAA incorporation efficiencies with SPSs were determined to be equal to or improved relative to efficiencies determined with DPSs. Click chemistry on yeast cells displaying ncAA-containing proteins was also shown to be feasible in both formats, although differences in reactivity between formats suggest the need for caution when using such approaches. Additionally, we investigated whether these reporters would support separation of yeast strains known to exhibit distinct ncAA incorporation efficiencies. Model sorts conducted with mixtures of two strains transformed with the same SPS or DPS led to enrichment of a strain known to support higher efficiency ncAA incorporation, suggesting that these reporters will be suitable for conducting screens for strains exhibiting enhanced ncAA incorporation efficiencies. Overall, our results confirm that SPSs are well-behaved in yeast and provide a convenient alternative to DPSs. In particular, SPSs are expected to be invaluable for conducting high-throughput investigations of the effects of genetic or genomic changes on ncAA incorporation efficiency and, more fundamentally, the eukaryotic translation apparatus.

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Evolution and Unprecedented Variants of the Mitochondrial Genetic Code in a Lineage of Green Algae

Genome Biology and Evolution ◽

10.1093/gbe/evz210 ◽

2019 ◽

Vol 11 (10) ◽

pp. 2992-3007 ◽

Cited By ~ 7

Author(s):

David Žihala ◽

Marek Eliáš

Keyword(s):

Genetic Code ◽

Protein Sequences ◽

Dynamic Evolution ◽

Translation System ◽

Release Factor ◽

Standard Genetic Code ◽

Green Algal ◽

Mitochondrial Genetic Code ◽

Species Analysis ◽

Direct Support

Abstract Mitochondria of diverse eukaryotes have evolved various departures from the standard genetic code, but the breadth of possible modifications and their phylogenetic distribution are known only incompletely. Furthermore, it is possible that some codon reassignments in previously sequenced mitogenomes have been missed, resulting in inaccurate protein sequences in databases. Here we show, considering the distribution of codons at conserved amino acid positions in mitogenome-encoded proteins, that mitochondria of the green algal order Sphaeropleales exhibit a diversity of codon reassignments, including previously missed ones and some that are unprecedented in any translation system examined so far, necessitating redefinition of existing translation tables and creating at least seven new ones. We resolve a previous controversy concerning the meaning the UAG codon in Hydrodictyaceae, which beyond any doubt encodes alanine. We further demonstrate that AGG, sometimes together with AGA, encodes alanine instead of arginine in diverse sphaeroplealeans. Further newly detected changes include Arg-to-Met reassignment of the AGG codon and Arg-to-Leu reassignment of the CGG codon in particular species. Analysis of tRNAs specified by sphaeroplealean mitogenomes provides direct support for and molecular underpinning of the proposed reassignments. Furthermore, we point to unique mutations in the mitochondrial release factor mtRF1a that correlate with changes in the use of termination codons in Sphaeropleales, including the two independent stop-to-sense UAG reassignments, the reintroduction of UGA in some Scenedesmaceae, and the sense-to-stop reassignment of UCA widespread in the group. Codon disappearance seems to be the main drive of the dynamic evolution of the mitochondrial genetic code in Sphaeropleales.

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A Bias in the Reading of the Genetic Code of Escherichia coli is a Characteristic for Genes that Specify Stress-induced MazF-mediated Proteins

Current Genomics ◽

10.2174/1389202921999200606215305 ◽

2020 ◽

Vol 21 (4) ◽

pp. 311-318

Author(s):

Akanksha Nigam ◽

Adi Oron-Gottesman ◽

Hanna Engelberg-Kulka

Keyword(s):

Escherichia Coli ◽

Genetic Code ◽

Translation System ◽

Translation Machinery ◽

E Coli

Background: Escherichia coli (E. coli) mazEF, a stress-induced toxin-antitoxin (TA) system, has been studied extensively. The MazF toxin is an endoribonuclease that cleaves RNAs at ACA sites. Thereby, under stress, the induced MazF generates a Stress-induced Translation Machinery (STM), composed of MazF processed mRNAs and selective ribosomes that specifically translate the processed mRNAs. Materials and Methods: Escherichia coli (E. coli) mazEF, a stress-induced toxin-antitoxin (TA) system, has been studied extensively. The MazF toxin is an endoribonuclease that cleaves RNAs at ACA sites. Thereby, under stress, the induced MazF generates a Stress-induced Translation Machinery (STM), composed of MazF processed mRNAs and selective ribosomes that specifically translate the processed mRNAs. Results: Here it is reported that for most of the E. coli proteins mediated by stress-induced MazF, the ACA threonine codon in their mRNAs is not in-frame but rather out-of-frame; in these same RNAs, the three synonymous threonine codons, ACG, ACU, and ACC, are in-frame. In contrast, for proteins translated by the canonical translation system, in the majority of mRNAs, the ACA codon is located in-frame. Conclusion: The described bias in the genetic code is a characteristic of E. coli genes specifying for stress-induced MazF-mediated proteins.

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