Instructions for the synthesis of unearthly proteins were written in four-letter words
For many years, scientists have been trying to develop a system that would allow embedding so-called artificial (structurally modified) amino acids in protein sequences that differ from natural analogues in physico-chemical and biological properties. By changing the genetic code and learning how to introduce artificial amino acids into proteins, scientists would be able to better understand the essence of natural cellular processes, as well as develop artificial ones based on natural biological systems that would have fundamentally new properties.
Previously, scientists have managed to achieve some success in this direction. As you know, the unit of the genetic code is a sequence of three nucleotides – a triplet or codon encoding a specific amino acid. Since there are only 4 different nucleotides: adenine (A), thymine (T) (uracil, U corresponds to it in RNA), cytosine (C) and guanine (G), there can be 4 3 = 64 possible combinations of triplets. However, there are only 22 natural amino acids, and 64 variants of triplets. This phenomenon is explained by the so–called degeneracy of the genetic code - one amino acid can be encoded by 4 variants of triplets differing by the last nucleotide (for example, valine can be encoded by GUU, GUC, GUA and GUG). The signal for stopping the synthesis of the polypeptide chain for ribosomes is 3 stop codons: UAA (ochre-), UAG (amber-) or UGA (opal-codon). Scientists have learned how to embed artificial non-natural (modified) amino acids into a natural protein chain using an amber codon: they changed the tRNA corresponding to the amber codon and created several such tRNAs connected to various modified amino acids, which allowed natural ribosomes to embed artificial amino acids into natural polypeptide chains. One such codon is clearly not enough for the synthesis of a completely non-natural protein. To do this, it is necessary to create a universal system for encoding and embedding non-natural amino acids in the polypeptide chain.
A group of scientists working under the guidance of synthetic biology specialist Jason Chin from the University of Cambridge (UK) have developed a system capable of recognizing codons from not three, but four nucleotides. Such a system will allow to genetically encode up to 256 (4 4) different modified amino acids.
Natural ribosomes that translate triplets into one of the 22 amino acids are not able to recognize quadruplets. And changing natural ribosomes in such a way that they could read the quadruplets and embed non-natural amino acids into protein chains would lead to disruption of physiological cellular processes, and possibly to cell death.
The researchers solved this problem as follows: they developed a new ribosome, the so-called orthogonal ribosome, changing its site responsible for recognizing the mRNA sequence, and also developed a special mRNA containing a sequence corresponding to the recognition site in the orthogonal ribosome. This approach allows the orthogonal ribosome to specifically recognize and translate the modified mRNA in the cell without affecting cellular mRNAs and natural ribosomes.
Scientists have demonstrated the possibility of simultaneous and independent functioning of mutant orthogonal and natural ribosomes in living cells: they introduced orthogonal ribosomes and a construct encoding the antibiotic resistance gene in a quadruplex form into bacterial cells and grew these cells in an environment containing an antibiotic encoded in 4-nucleotide codons. Cells containing orthogonal ribosomes, which successfully coped with the task of recognizing and synthesizing protein by quadruplex codons, survived even in conditions of high concentrations of the antibiotic, because they contained newly synthesized proteins that utilize this antibiotic.
Orthogonal ribosomes are also capable of translating triplets, but preference is given to quadruplets. For this reason, orthogonal ribosomes are able to embed natural and modified amino acids encoded by the amber codon into growing polypeptide chains.
By mastering the technique of translation of quadruplet codons, scientists can synthesize proteins containing several modified amino acids. Chin's group managed to synthesize using an orthogonal ribosome a protein containing 2 modified amino acids, one of which was encoded by an amber codon, and the other by a quadruplet.
The demonstration of the possibility of translation in living cells of quadruplets into proteins containing artificial amino acids opens a new era in synthetic biology.
The article by Heinz Neumann, Adrian L. Slusarczyk and Jason W. Chin "De Novo Generation of Mutually Orthogonal Aminoacyl-tRNA Synthetase/tRNA Pairs", dedicated to the development of a method for recognizing a codon and matching a modified amino acid to it, was published in the Journal of American Chemical Society on February 1 this year.
Daria Chervyakova
Portal "Eternal youth" http://vechnayamolodost.ru based on the materials of The Scientist: Genetic coding revamp17.02.2010