Expanded genetic code
Encyclopedia
An expanded genetic code refers to an artificially modified genetic code
in which one or more specific codons have been allocated to encode an amino acid
which is not among the twenty/twenty-two found in nature.
s. Transfer RNA
s (tRNA) are used as keys to decode the RNA into its equivalent polypeptide. The tRNA recognises a specific three nucleotide codon thanks to a complementary sequence called the anticodon on one of its loops. Each three nucleotide codon is translated into one amino acid. There is at least one tRNA for any codon. If there are more than one, they code for the same amino acid. Many tRNAs are compatible with several codons. The encoding of a codon to its amino acid is a result of the aminoacyl tRNA synthetase
which adds the aminoacyl group to its allocated tRNA. The aminoacyl tRNA synthetase often does not recognise the anticodon, but another part of the tRNA, meaning that if the anticodon were to be mutated the encoding of that amino acid would change to a new codon.
set (called the orthogonal set when including the codon) must not crosstalk with the endogenous tRNA and synthetase sets, while still being functionally compatible with the ribosome and other components of the translation apparatus. The tRNA synthetase pair is taken from a distant organism, generally from a different domain, and the active site of the synthetase is modified to accept the non-natural amino acid.
The possibility of reassigning codons was realized by Normanly et al. in 1990 when a viable mutant strain of E. coli read through the amber (stop) codon. As a result the amber codon became the choice codon to be assigned a novel amino acid. Later, in the Schultz lab the tRNATyr/tyrosyl-tRNA synthetase (TyrRS) from Methanococcus jannaschii was used to introduce a tyrosine instead of STOP, the default value of the amber codon. As mentioned, this was possible because of the differences between the endogenous bacterial synthases and the orthologous archeal synthase which do not recognise each other.
Selection involves multiple rounds of a two-step process, where the plasmid is transferred into cells expressing chloramphenicol acetyl transferase with a premature amber codon. In the presence of toxic chloramphenicol and the non-natural amino acid, the surviving cells will have overridden the amber codon using the orthogonal tRNA aminoacylated with either the standard amino acids or the non-natural one. To remove the former, the plasmid is inserted into cells with a barnase gene (toxic) with a premature amber codon but without the non-natural amino acid, removing all the orthogonal synthases which do not specifically recognize the non-natural amino acid.
In addition to the recoding of the tRNA to a different codon, they can be mutated to recognize a four base codon, allowing additional free coding options.
The non natural amino acid, as a result, introduces diverse physicochemical and biological properties in order to be used as a tool to explore protein structure
and function or to create novel or enhanced protein for practical purposes.
and the -amino group of lysine. Also, an expanded genetic code allows modifications to be carried out in vivo.
The ability to site-specifically direct lab-synthesized chemical moieties into proteins allows many types of studies which would otherwise be extremely difficult.
An example of the possible application for this method is the biomedical where "chemical warheads" can be added to protein which target specific cellular components.
Genetic code
The genetic code is the set of rules by which information encoded in genetic material is translated into proteins by living cells....
in which one or more specific codons have been allocated to encode an amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
which is not among the twenty/twenty-two found in nature.
Background
The translation is catalysed by ribosomeRibosome
A ribosome is a component of cells that assembles the twenty specific amino acid molecules to form the particular protein molecule determined by the nucleotide sequence of an RNA molecule....
s. Transfer RNA
Transfer RNA
Transfer RNA is an adaptor molecule composed of RNA, typically 73 to 93 nucleotides in length, that is used in biology to bridge the three-letter genetic code in messenger RNA with the twenty-letter code of amino acids in proteins. The role of tRNA as an adaptor is best understood by...
s (tRNA) are used as keys to decode the RNA into its equivalent polypeptide. The tRNA recognises a specific three nucleotide codon thanks to a complementary sequence called the anticodon on one of its loops. Each three nucleotide codon is translated into one amino acid. There is at least one tRNA for any codon. If there are more than one, they code for the same amino acid. Many tRNAs are compatible with several codons. The encoding of a codon to its amino acid is a result of the aminoacyl tRNA synthetase
Aminoacyl tRNA synthetase
An aminoacyl tRNA synthetase is an enzyme that catalyzes the esterification of a specific amino acid or its precursor to one of all its compatible cognate tRNAs to form an aminoacyl-tRNA. This is sometimes called "charging" the tRNA with the amino acid...
which adds the aminoacyl group to its allocated tRNA. The aminoacyl tRNA synthetase often does not recognise the anticodon, but another part of the tRNA, meaning that if the anticodon were to be mutated the encoding of that amino acid would change to a new codon.
Introduction
For successful translation of a novel amino acid, the codon to which the amino acid is reassigned must be free or unfavoured and the novel tRNA and synthetaseAminoacyl tRNA synthetase
An aminoacyl tRNA synthetase is an enzyme that catalyzes the esterification of a specific amino acid or its precursor to one of all its compatible cognate tRNAs to form an aminoacyl-tRNA. This is sometimes called "charging" the tRNA with the amino acid...
set (called the orthogonal set when including the codon) must not crosstalk with the endogenous tRNA and synthetase sets, while still being functionally compatible with the ribosome and other components of the translation apparatus. The tRNA synthetase pair is taken from a distant organism, generally from a different domain, and the active site of the synthetase is modified to accept the non-natural amino acid.
The possibility of reassigning codons was realized by Normanly et al. in 1990 when a viable mutant strain of E. coli read through the amber (stop) codon. As a result the amber codon became the choice codon to be assigned a novel amino acid. Later, in the Schultz lab the tRNATyr/tyrosyl-tRNA synthetase (TyrRS) from Methanococcus jannaschii was used to introduce a tyrosine instead of STOP, the default value of the amber codon. As mentioned, this was possible because of the differences between the endogenous bacterial synthases and the orthologous archeal synthase which do not recognise each other.
Directed evolution
This orthologous set can then be mutated and screened through directed evolution to accept a different, even novel, amino acid. Mutations to the plasmid containing the pair can be introduced by error-prone PCR or through degenerate primers for the synthetase's active site.Selection involves multiple rounds of a two-step process, where the plasmid is transferred into cells expressing chloramphenicol acetyl transferase with a premature amber codon. In the presence of toxic chloramphenicol and the non-natural amino acid, the surviving cells will have overridden the amber codon using the orthogonal tRNA aminoacylated with either the standard amino acids or the non-natural one. To remove the former, the plasmid is inserted into cells with a barnase gene (toxic) with a premature amber codon but without the non-natural amino acid, removing all the orthogonal synthases which do not specifically recognize the non-natural amino acid.
In addition to the recoding of the tRNA to a different codon, they can be mutated to recognize a four base codon, allowing additional free coding options.
The non natural amino acid, as a result, introduces diverse physicochemical and biological properties in order to be used as a tool to explore protein structure
Protein structure
Proteins are an important class of biological macromolecules present in all organisms. Proteins are polymers of amino acids. Classified by their physical size, proteins are nanoparticles . Each protein polymer – also known as a polypeptide – consists of a sequence formed from 20 possible L-α-amino...
and function or to create novel or enhanced protein for practical purposes.
Diversity
The orthogonal pairs of synthase and tRNA which work for one organism may not work for another as the synthase may mis-aminoacylate endogenous tRNAs or the tRNA be mis-aminoacylated itself by an endogenous synthase. As a result the sets created to date differ between organisms.orthogonal sets in E. coli
- tRNATyr-TyrRS pair from the archaeon Methanococcus jannaschii
- tRNALys–LysRS pair from the archaeon Pyrococcus horikoshii
- tRNAGlu–GluRS pair from Methanosarcina mazei
- leucyl-tRNA synthetase from Methanobacterium thermoautotrophicum and a mutant leucyl tRNA derived from Halobacterium sp
orthogonal sets in yeast
- tRNATyr-TyrRS pair from Escherichia coli
- tRNALeu–LeuRS pair from Escherichia coli
- tRNAiMet from human and GlnRS from Escherichia coli
orthogonal sets in mammalian cells
- tRNATyr-TyrRS pair from Bacillus stearothermophilus
- modified tRNATrp-TrpRS pair from Bacillus subtilis trp
- tRNALeu–LeuRS pair from Escherichia coli
Protein studies
With an expanded genetic code, the unnatural amino acid can be genetically directed to any chosen site in the protein of interest. The high efficiency and fidelity allows a better control of the placement of the modification compared to modifying the protein post-translationally, which generally will target all amino acids of the same type, such as the thiol group of cysteineCysteine
Cysteine is an α-amino acid with the chemical formula HO2CCHCH2SH. It is a non-essential amino acid, which means that it is biosynthesized in humans. Its codons are UGU and UGC. The side chain on cysteine is thiol, which is polar and thus cysteine is usually classified as a hydrophilic amino acid...
and the -amino group of lysine. Also, an expanded genetic code allows modifications to be carried out in vivo.
The ability to site-specifically direct lab-synthesized chemical moieties into proteins allows many types of studies which would otherwise be extremely difficult.
- Probing Protein Structure and Function: by using amino acids with slightly different size such as o-Methyltyrosine or dansylalanine instead of tyrosine, and by inserting genetically coded reporter moieties (color-changing and/or spin-active) into selected protein sites, chemical information about the protein's structure and function can be measured.
- Identifying and Regulating Protein Activity: by using photocaged aminoacids, protein function can be "switched" on or off by illuminating the organism.
- Changing the mode of action of a protein: one can start with the gene for a protein which binds a certain sequence of DNA, and, by inserting a chemically active amino acid into the binding site, convert it to a protein which cuts the DNA, rather than binding it.
- Improving immunogenicity and overcoming self-tolerance: by replacing strategically chosen tyrosines with p-nitro phenylalanine, a tolerated self-protein can be made immunogenic.
An example of the possible application for this method is the biomedical where "chemical warheads" can be added to protein which target specific cellular components.
See also
- synthetic biologySynthetic biologySynthetic biology is a new area of biological research that combines science and engineering. It encompasses a variety of different approaches, methodologies, and disciplines with a variety of definitions...
- Novel base-pairsNucleic acid analoguesNucleic acid analogues are compounds structurally similar to naturally occurring RNA and DNA, used in medicine and in molecular biology research....
- Directed evolutionDirected evolutionthumb|250px|right|An example of a possible round to evolve a protein based fluorescent sensor for a specific analyte using two consecutive FACS sortings...
- BioBrickBioBrickBioBrick standard biological parts are DNA sequences of defined structure and function; they share a common interface and are designed to be composed and incorporated into living cells such as E. coli to construct new biological systems. BioBrick parts represent an effort to introduce the...
- IGEMIGEMThe International Genetically Engineered Machine competition is a worldwide Synthetic Biology competition aimed at undergraduate university students.- Competition details :...
- bioengineering
- protein labelling
- protein methodsProtein methodsProtein methods are the techniques used to study proteins.There are genetic methods for studying proteins, methods for detecting proteins, methods for isolating and purifying proteins and other methods for characterizing the structure and function of proteins, often requiring that the protein first...