Expansion of the genetic code: site-directed p-fluoro-phenylalanine incorporation in Escherichia coli.

Site-directed incorporation of the amino acid analogue p-fluoro-phenylalanine (p-F-Phe) was achieved in Escherichia coli. A yeast suppressor tRNA(Phe)amber/phenylalanyl-tRNA synthetase pair was expressed in an analogue-resistant E. coli strain to direct analogue incorporation at a programmed amber stop codon in the DHFR marker protein. The programmed position was translated to 64-75% as p-F-Phe and the remainder as phenylalanine and lysine. Depending on the expression conditions, the p-F-Phe incorporation was 11-21-fold higher at the programmed position than the background incorporation at phenylalanine codons, showing high specificity of analogue incorporation. Protein expression yields of 8-12 mg/L of culture, corresponding to about two thirds of the expression level of the wild-type DHFR protein, are sufficient to provide fluorinated proteins suitable for 19F-NMR spectroscopy and other sample-intensive methods. The use of a nonessential "21st" tRNA/synthetase pair will permit incorporation of a wide range of analogues, once the synthetase specificity has been modified accordingly.     

The genetic code and the origin of life

The problem of the origin of life understandably counts as one of the most exciting questions in the natural sciences, but in spite of almost endless speculation on this subject, it is still far from its final solution. The complexity of the functional correlation between recent nucleic acids and proteins can e.g. give rise to the assumption that the genetic code (and life) could not originate on the Earth. It was Portelli (1975) who published the hypothesis that the genetic code could not originate during the history of the Earth. In his opinion the recent genetic code represents the informational message transmitted by living systems of the previous cycle of the Universe. Here however, we defend the existence of a certain strategy in the syntheses of the genetic code during the history of the Earth. The strategy of correlation between amino acid and nucleotide polymers made an increasing velocity of the chemical evolution possible, that is, it increased the velocity of formation of the genetic code. Thus, life with the recent genetic code could originate on the Earth within the present cycle of the Universe.Present address: Institute for Pharmacy and Biochemistry, 533 51 Pardubice, Czechoslovakia.     

New opportunities for genetic code expansion in synthetic yeast

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Origins of gene, genetic code, protein and life: comprehensive view of life systems from a GNC-SNS primitive genetic code hypothesis

We have investigated the origin of genes, the genetic code, proteins and life using six indices (hydropathy, α-helix, β-sheet and β-turn formabilities, acidic amino acid content and basic amino acid content) necessary for appropriate three-dimensional structure formation of globular proteins. From the analysis of microbial genes, we have concluded that newly-born genes are products of nonstop frames (NSF) on antisense strands of microbial GC-rich genes [GC-NSF(a)] and from SNS repeating sequences [(SNS)_n] similar to the GC-NSF(a) (S and N mean G or C and either of four bases, respectively). We have also proposed that the universal genetic code used by most organisms on the earth presently could be derived from a GNC-SNS primitive genetic code. We have further presented the [GADV]-protein world hypothesis of the origin of life as well as a hypothesis of protein production, suggesting that proteins were originally produced by random peptide formation of amino acids restricted in specific amino acid compositions termed as GNC-, SNS and GC-NSF(a)-0th order structures of proteins. The [GADV]-protein world hypothesis is primarily derived from the GNC-primitive genetic code hypothesis. It is also expected that basic properties of extant genes and proteins could be revealed by considerations based on the scenario with four stages This review is a modified English version of the paper, which was written in Japanese and published inViva Origino 2001 29 66–85.     

An RNA-amino acid complex and the origin of the genetic code

The group I RNAs, of which the Tetrahymena ribosomal RNA intron is the most investigated example, catalyze their own splicing reactions. Splicing is initiated at a conserved site on the RNA that facilitates attack by exogenous guanosine (or its nucleotides) on the exon-intron junction. The guanosine site in the RNA's catalytic center also binds arginine, and is quite selective for the arginine side chain. This amino acid-RNA interaction is stereoselective, and L-arginine is preferred. Immediately at the site at which arginine binds there is one of only four RNA triplets in 92 group I RNA sequences: AGA/G and CGA/G. Thus the arginine contact site is within any of four different codons for arginine. Mutation of the conserved G in the middle of the triplet decreases affinity for the amino acid, showing that binding is sequence-specific. A pathway for the origin of the genetic code for arginine is suggested, based on the existence and properties of this sequence-specific, amino acid-specific RNA complex. The existence of a proto-ribosome related to the group I RNAs seems the most likely hypothesis. This notion is used to distinguish three periods in the development of the code. Restrained and exuberant hypotheses about the origin of the genetic code are distinguished, and some objections to these hypotheses are considered.     

A concept of amino acid archaeorelation: Origin of life and the genetic code

Using certain assumptions, an attempt has been made to obtain a quantitative evaluation of the similarity of the bulk amino acid compositions of various organisms and of the quantitative ratios of amino acids in the products of the presumable prebiological ructions and in the natural proteins. The results of comparison between the molar ratios of amino acids in synthesized products and in natural proteins and the quantitative distribution of triplets among amino acids in the existing genetic code are given. Proceeding from the correlations found and the data reported in literature, a concept has been suggested amounting, in essence, to the assertion that the structure of the genetic code (the quantitative distribution of the triplets) is in a certain way determined by the pre-existing, primary ratio (archaeorelation) of amino acids.     

Different life strategies in genetic backgrounds of the Saccharomyces cerevisiae yeast cells

Changes in the natural environment require an organism to make constant adaptations enabling efficient use of environmental resources and ensuring its success in competition with other organisms. Such adaptations are expressed through various life strategies, largely determined by the rate of consumption and use of available resources, affecting the life-history traits and the related trade-offs. Allocation of available resources must take into consideration the costs of cell maintenance as well as reproduction. Given that carbon metabolism plays a crucial role in resource allocation, yeast living in different ecological niches show various life-history traits. There are a lot of data about life-history strategies in yeast living in various ecological niches; however, the question is whether different life strategies will be noted for yeast strains growing under strictly controlled conditions. Our studies based on three laboratory yeast strains representing different genetic backgrounds show that each of these strains has specified life strategies which are mainly determined by the glucose uptake rate and its intracellular usage. These results suggest that specific life strategies and related differences in the physiological and metabolic parameters of the cell are the key aspects that may explain various features of cells from different yeast strains, either industrial or laboratory.     

A complex genetic locus controlling purine nucleotide biosynthesis in yeast

Summary Mutants of the genes pur1 to pur6 excrete purine when in combination with the allele su-pur ⁺ and are resistant to growth inhibition by 8-azaadenine (8-AzAd) and 8-azaguanine (8-AzGu). In combination with su-pur, which suppresses purine excretion, pur1 and pur2 are analogue sensitive; pur3 is slightly resistant to 8-AzAd; pur4 is slightly resistant to both analogues and pur5 is completely resistant to 8-AzGu. Crosses of the pur mutants to dap, which causes sensitivity to 2,6-diaminopurine (2,6-DAP), guanine and 6-mercaptopurine (6-MP), show that dap also suppresses purine excretion and is closely linked to pur6. In combination with dap, pur1 and pur3 are analogue sensitive; pur4 is hypersensitive to guanine but resistant to 6-MP; pur5 is resistant to 2,6-DAP and guanine whilst pur2 is hypersensitive to all three compounds.The gene slw, which, like pur2, potentiates the effects of dap, also suppresses purine excretion but is not linked to any of the pur genes. The diploid slw/pur3 excretes purine.Tests for functional allelism were carried out on the closely linked genotypes su-pur ⁺, su-pur, dap, pur6, PUR6 and ade4. The results of these tests indicate that all six genotypes are functionally allelic. It is suggested that a molecular complex of the products of pur1, pur3, pur4, pur6 and slw is involved in the control of purine nucleotide biosynthesis in yeast.     

Mistranslation of the genetic code

During mRNA decoding at the ribosome, deviations from stringent codon identity, or “mistranslation,” are generally deleterious and infrequent. Observations of organisms that decode some codons ambiguously, and the discovery of a compensatory increase in mistranslation frequency to combat environmental stress have changed the way we view “errors” in decoding. Modern tools for the study of the frequency and phenotypic effects of mistranslation can provide quantitative and sensitive measurements of decoding errors that were previously inaccessible. Mistranslation with non-protein amino acids, in particular, is an enticing prospect for new drug therapies and the study of molecular evolution.     

Working of the genetic code

The genetic code is used differently by different kinds of species. Each type of genome has a particular coding strategy, that is, choices among degenerate bases are consistently similar for all genes therein. This uniformity in the selection between degenerate bases within each taxonomic group has been discovered by applying new methods to the study of coding variability. It is now possible to calculate relative distances between genomes, or genome types, based on use of the codon catalog by the mRNAs therein.     

On malleability in the genetic code

To explain now-numerous cases of codon reassignment (departure from the “universal” code), we suggest a pathway in which the transformed codon is temporarily ambiguous. All the unusual tRNA activities required have been demonstrated. In addition, the repetitive use of certain reassignments, the phylogenetic distribution of reassignments, and the properties of present-day reassigned tRNAs are each consistent with evolution of the code via an ambiguous translational intermediate. Correspondence to: M. Yarns