Isolation of a rat mitochondrial release factor. Accommodation of the changed genetic code for termination

A single release factor has been isolated and partially purified from rat mitochondria. It requires ethanol in addition to the specific termination codon when assayed in a heterologous system with Escherichia coli ribosomes. The factor recognizes the codons UAA and UAG but not UGA, and therefore it has been designated mtRF-1. A factor of the bacterial RF-2 type, which in E. coli recognizes UGA, or of the mammalian type, which recognizes all three termination codons, has not been detected in mitochondria. The absence of a factor responding to UGA accommodates the use of this codon as a signal for tryptophan in the rat mitochondrial genetic code. The mtRF-1 could translate all of the known termination codons in the rat mitochondrial genome. It does not respond to AGG and AGA which in bovine and human mitochondrial DNA code for termination but which in rat mitochondria may not code for either an amino acid or for termination.     

Accurate translation of the genetic code depends on tRNA modified nucleosides

Transfer RNA molecules translate the genetic code by recognizing cognate mRNA codons during protein synthesis. The anticodon wobble at position 34 and the nucleotide immediately 3' to the anticodon triplet at position 37 display a large diversity of modified nucleosides in the tRNAs of all organisms. We show that tRNA species translating 2-fold degenerate codons require a modified U(34) to enable recognition of their cognate codons ending in A or G but restrict reading of noncognate or near-cognate codons ending in U and C that specify a different amino acid. In particular, the nucleoside modifications 2-thiouridine at position 34 (s(2)U(34)), 5-methylaminomethyluridine at position 34 (mnm(5)U(34)), and 6-threonylcarbamoyladenosine at position 37 (t(6)A(37)) were essential for Watson-Crick (AAA) and wobble (AAG) cognate codon recognition by tRNA(UUU)(Lys) at the ribosomal aminoacyl and peptidyl sites but did not enable the recognition of the asparagine codons (AAU and AAC). We conclude that modified nucleosides evolved to modulate an anticodon domain structure necessary for many tRNA species to accurately translate the genetic code.     

Using a quadruplet codon to expand the genetic code of an animal

Genetic code expansion in multicellular organisms is currently limited to the use of repurposed amber stop codons. Here, we introduce a system for the use of quadruplet codons to direct incorporation of non-canonical amino acids in vivo in an animal, the nematode worm Caenorhabditis elegans. We develop hybrid pyrrolysyl tRNA variants to incorporate non-canonical amino acids in response to the quadruplet codon UAGA. We demonstrate the efficiency of the quadruplet decoding system by incorporating photocaged amino acids into two proteins widely used as genetic tools. We use photocaged lysine to express photocaged Cre recombinase for the optical control of gene expression and photocaged cysteine to express photo-activatable caspase for light inducible cell ablation. Our approach will facilitate the routine adoption of quadruplet decoding for genetic code expansion in eukaryotic cells and multicellular organisms.     

Creation,Optimization, and Use of Semi-Synthetic Organisms that Store and Retrieve Increased Genetic Information

With few exceptions, natural proteins are built from only 20 canonical (proteogenic) amino acids which limits the functionality and accordingly the properties they can possess. Genetic code expansion, i.e. the creation of codons and the machinery needed to assign them to non-canonical amino acids (ncAAs), promises to enable the discovery of proteins with novel properties that are otherwise difficult or impossible to obtain. One approach to expanding the genetic code is to expand the genetic alphabet via the development of unnatural nucleotides that pair to form an unnatural base pair (UBP). Semi-synthetic organisms (SSOs), i.e. organisms that stably maintain the UBP, transcribe its component nucleotides into RNA, and use it to translate proteins, would have available to them new codons and the anticodons needed to assign them to ncAAs. This review summarizes the development of a family of UBPs, their use to create SSOs, and the optimization and application of the SSOs to produce candidate therapeutic proteins with improved properties that are now undergoing evaluation in clinical trials.     

Structuring of the genetic code took place at acidic pH

I have observed that in multiple regression the number of codons specifying amino acids in the genetic code is positively correlated with the isoelectric point of amino acids and their molecular weight. Therefore basic amino acids are, on average, codified in the genetic code by a larger number of codons, which seems to imply that the genetic code originated in an acidic 'intracellular' environment. Moreover, I compare the proteins from Picrophilus torridus and Thermoplasma volcanium, which have different intracellular pH and I define the ranks of acidophily for the amino acids. A simple index of acidophily (AI), which can be easily obtained from acidophily ranks, can be associated to any protein and, therefore, can also be associated to the genetic code if the number of synonymous codons attributed to the amino acids in the code is assumed to be the frequency with which the amino acids appeared in ancestral proteins. Finally, the sampling of the variable AI among organisms having an intracellular pH less than or equal to 6.6 and those having a non-acidic intracellular pH leads to the conclusion that the value of the genetic code's AI is not typical of proteins of the latter organisms. As the genetic code's AI value is also statistically not different from that of proteins of the organisms having an acidic intracellular pH, this supports the hypothesis that the structuring of the genetic code took place in acidic pH conditions.     

Genetic Code Expansion Through Quadruplet Codon Decoding

Noncanonical amino acid mutagenesis has emerged as a powerful tool for the study of protein structure and function. While triplet nonsense codons, especially the amber codon, have been widely employed, quadruplet codons have attracted attention for the potential of creating additional blank codons for noncanonical amino acids mutagenesis. In this review, we discuss methodologies and applications of quadruplet codon decoding in genetic code expansion both in vitro and in vivo.     

Pyrrolysyl-tRNA synthetase: An ordinary enzyme but an outstanding genetic code expansion tool

The genetic incorporation of the 22nd proteinogenic amino acid, pyrrolysine (Pyl) at amber codon is achieved by the action of pyrrolysyl-tRNA synthetase (PylRS) together with its cognate tRNA^Pyl. Unlike most aminoacyl-tRNA synthetases, PylRS displays high substrate side chain promiscuity, low selectivity toward its substrate α-amine, and low selectivity toward the anticodon of tRNA^Pyl. These unique but ordinary features of PylRS as an aminoacyl-tRNA synthetase allow the Pyl incorporation machinery to be easily engineered for the genetic incorporation of more than 100 non-canonical amino acids (NCAAs) or α-hydroxy acids into proteins at amber codon and the reassignment of other codons such as ochre UAA, opal UGA, and four-base AGGA codons to code NCAAs.     

In vitro selection for sense codon suppression

The universal genetic code links the 20 naturally occurring amino acids to the 61 sense codons. Previously, the UAG amber stop codon (a nonsense codon) has been used as a blank in the code to insert natural and unnatural amino acids via nonsense suppression. We have developed a selection methodology to investigate whether the unnatural amino acid biocytin could be incorporated into an mRNA display library at sense codons. In these experiments we probed a single randomized NNN codon with a library of 16 orthogonal, biocytin-acylated tRNAs. In vitro selection for efficient incorporation of the unnatural amino acid resulted in templates containing the GUA codon at the randomized position. This sense suppression occurs via Watson-Crick pairing with similar efficiency to UAG-mediated nonsense suppression. These experiments suggest that sense codon suppression is a viable means to expand the chemical and functional diversity of the genetic code.     

Neurospora crassa supersuppressor mutants are amber codon-specific

Neurospora crassa has 10 mapped supersuppressor (ssu) genes. In vivo studies indicate that they suppress amber (UAG) premature termination mutations but the spectrum of their functions remains to be elucidated. We examined seven ssu strains (ssu-1, -2, -3, -4, -5, -9, and -10) using cell-free translation extracts. We tested suppression by requiring it to produce firefly luciferase from a reading frame containing premature UAA, UGA, or UAG terminators. All mutants except ssu-3 suppressed UAG codons. Maximal UAG suppression ranged from 15% to 30% relative to controls containing sense codons at the corresponding position. Production from constructs containing UAA or UGA was 1-2%, similar to levels observed with all nonsense codons in wild-type and ssu-3 extracts. UAG suppression was also seen using [35S]Met to radiolabel polypeptides. Suppression enabled ribosomes to continue translation elongation as determined using the toeprint assay. tRNA from supersuppressors showed suppressor activity when added to wild-type extracts. Thus, these supersuppressors produce amber suppressor tRNA.     

Evolutionary changes in the genetic code

The genetic code has been influenced by directional mutation pressure affecting the base composition of DNA, sometimes in the direction of increased GC content and at other times, in the direction of AT. Such pressure led to changes in species-specific usages of codons and tRNA anticodons, and also in amino acid assignments of codons in mitochondria and in several intact organisms. These code changes are probably recent evolutionary events. The genetic code is not 'frozen', but instead it is still evolving.     

Evolution of the Genetic Code by Incorporation of Amino Acids that Improved or Changed Protein Function

Fifty years have passed since the genetic code was deciphered, but how the genetic code came into being has not been satisfactorily addressed. It is now widely accepted that the earliest genetic code did not encode all 20 amino acids found in the universal genetic code as some amino acids have complex biosynthetic pathways and likely were not available from the environment. Therefore, the genetic code evolved as pathways for synthesis of new amino acids became available. One hypothesis proposes that early in the evolution of the genetic code four amino acids—valine, alanine, aspartic acid, and glycine—were coded by GNC codons (N = any base) with the remaining codons being nonsense codons. The other sixteen amino acids were subsequently added to the genetic code by changing nonsense codons into sense codons for these amino acids. Improvement in protein function is presumed to be the driving force behind the evolution of the code, but how improved function was achieved by adding amino acids has not been examined. Based on an analysis of amino acid function in proteins, an evolutionary mechanism for expansion of the genetic code is described in which individual coded amino acids were replaced by new amino acids that used nonsense codons differing by one base change from the sense codons previously used. The improved or altered protein function afforded by the changes in amino acid function provided the selective advantage underlying the expansion of the genetic code. Analysis of amino acid properties and functions explains why amino acids are found in their respective positions in the genetic code.     

The genetic code of the fungal CTG clade

Genetic code alterations discovered over the last 40 years in bacteria and eukaryotes invalidate the hypothesis that the code is universal and frozen. Mitochondria of various yeast species translate the UGA stop codon as tryptophan (Trp) and leucine (Leu) CUN codons (N = any nucleotide) as threonine (Thr) and fungal CTG clade species reassigned Leu CUG codons to serine and translate them ambiguously in their cytoplasms. This unique sense-to-sense genetic code alteration is mediated by a Ser-tRNA containing a Leu 5'-CAG-3'anticodon (ser-tRNA(CAG)), which is recognized and charged with Ser (~97%) by the seryl-tRNA synthetase (SerRS) and with Leu (~3%) by the leucyl-tRNA synthetase (LeuRS). This unusual tRNA appeared 272 ± 25 million years ago and had a profound impact on the evolution of the CTG clade species. Here, we review the most recent results and concepts arising from the study of this codon reassignment and we highlight how its study is changing our views of the evolution of the genetic code.     

The influence of the reading context upon the suppression of nonsense codons

Summary One of the basic assumptions of the current views of the genetic code is that the translation machinery reads the messenger RNA one nucleotide triplet codon at a time and that the meaning of a particular codon should not be effected by the surrounding nucleotide sequence (the reading context). Reexamination of existing data shows that this assumption does not hold for the case of suppression of the nonsense codons UAG (amber) and UAA (ochre).The efficiency of amino acid insertion in response to these nonsense codons appears to strongly depend on their location within the message. It is suggested that the translation machinery may interact with a nucleotide sequence longer than three nucleotides when involved in a chain termination reaction.     

Engineering Translation Components for Genetic Code Expansion

The expansion of the genetic code consisting of four bases and 20 amino acids into diverse building blocks has been an exciting topic in synthetic biology. Many biochemical components are involved in gene expression; therefore, adding a new component to the genetic code requires engineering many other components that interact with it. Genetic code expansion has advanced significantly for the last two decades with the engineering of several components involved in protein synthesis. These components include tRNA/aminoacyl-tRNA synthetase, new codons, ribosomes, and elongation factor Tu. In addition, biosynthesis and enhanced uptake of non-canonical amino acids have been attempted and have made meaningful progress. This review discusses the efforts to engineer these translation components, to improve the genetic code expansion technology.     

The evolutionary change of the genetic code as restricted by the anticodon and identity of transfer RNA

The discovery of non-universal genetic codes in several mitochondria and nuclear systems during the past ten years has necessitated a reconsideration of the concept that the genetic code is universal and frozen, as was once believed. Here, the flexibility of the relationship between codons and amino acids is discussed on the basis of the distribution of non-universal genetic codes in various organisms insofar as has been observed to date. Judging from the result of recent investigations into tRNA identity, it would appear that the non-participation of the anticodon in recognition by aminoacyl-tRNA synthetase has significantly influenced the variability of codons.     

Multiplexing mechanical and translational cues on genes

The genetic code gives precise instructions on how to translate codons into amino acids. Due to the degeneracy of the genetic code—18 out of 20 amino acids are encoded for by more than one codon—more information can be stored in a basepair sequence. Indeed, various types of additional information have been discussed in the literature, e.g., the positioning of nucleosomes along eukaryotic genomes and the modulation of the translating efficiency in ribosomes to influence cotranslational protein folding. The purpose of this study is to show that it is indeed possible to carry more than one additional layer of information on top of a gene. In particular, we show how much translation efficiency and nucleosome positioning can be adjusted simultaneously without changing the encoded protein. We achieve this by mapping genes on weighted graphs that contain all synonymous genes, and then finding shortest paths through these graphs. This enables us, for example, to readjust the disrupted translational efficiency profile after a gene has been introduced from one organism (e.g., human) into another (e.g., yeast) without greatly changing the nucleosome landscape intrinsically encoded by the DNA molecule.     

Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion

In vivo incorporation of unnatural amino acids by amber codon suppression is limited by release factor-1-mediated peptide chain termination. Orthogonal ribosome-mRNA pairs function in parallel with, but independent of, natural ribosomes and mRNAs. Here we show that an evolved orthogonal ribosome (ribo-X) improves tRNA(CUA)-dependent decoding of amber codons placed in orthogonal mRNA. By combining ribo-X, orthogonal mRNAs and orthogonal aminoacyl-tRNA synthetase/tRNA pairs in Escherichia coli, we increase the efficiency of site-specific unnatural amino acid incorporation from approximately 20% to >60% on a single amber codon and from <1% to >20% on two amber codons. We hypothesize that these increases result from a decreased functional interaction of the orthogonal ribosome with release factor-1. This technology should minimize the functional and phenotypic effects of truncated proteins in experiments that use unnatural amino acid incorporation to probe protein function in vivo.     

Mutation in the D arm enables a suppressor with a CUA anticodon to read both amber and ochre codons in Escherichia coli

Su9 of Escherichia coli differs from tRNATrp by only a G to A transition in the D arm, yet has an enhanced ability to translate UGA by an unusual C X A wobble pairing. In order to examine the effects of this mutation on translation of the complementary and wobble codons in vivo, we constructed the gene for an amber (UAG) suppressing variant of Su9, trpT179, by making the additional nucleotide change required for an amber suppressor anticodon. The resultant suppressor tRNA, Su79, is a very strong amber suppressor. Furthermore, the D arm mutation enables Su79 to suppress ochre (UAA) codons by C X A wobble pairing. These data demonstrate that the effect of the D arm mutation on wobble pairing is not restricted to a CCA anticodon. The effect extends to the CUA anticodon of Su79, thereby creating a new type of ochre suppressor. The new coding activity of Su79 cannot be explained by alterations in the level of aminoacylation, steady-state tRNA concentration, or nucleotide modification. The A24 mutation could permit unorthodox wobble pairings by generally enhancing tRNA efficiency at all codons or by altering codon specificity.