Biomaterial Database

Adaptation of an orthogonal archaeal leucyl-tRNA and synthetase pair for four-base, amber, and opal suppression. 2003

J Christopher Anderson, and Peter G Schultz

Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.

Recently, it has been shown that an amber suppressor tRNA/aminoacyl-tRNA synthetase pair derived from the tyrosyl-tRNA synthetase of Methanococcus jannaschii can be used to genetically encode unnatural amino acids in response to the amber nonsense codon, TAG. However, we have been unable to modify this pair to decode either the opal nonsense codon, TGA, or the four-base codon, AGGA, limiting us to a 21 amino acid code. To overcome this limitation, we have adapted a leucyl-tRNA synthetase from Methanobacterium thermoautotrophicum and leucyl tRNA derived from Halobacterium sp. NRC-1 as an orthogonal tRNA-synthetase pair in Escherichia coli to decode amber (TAG), opal (TGA), and four-base (AGGA) codons. To improve the efficiency and selectivity of the suppressor tRNA, extensive mutagenesis was performed on the anticodon loop and acceptor stem. The two most significant criteria required for an efficient amber orthogonal suppressor tRNA are a CU(X)XXXAA anticodon loop and the lack of noncanonical or mismatched base pairs in the stem regions. These changes afford only weak suppression of TGA and AGGA. However, this information together with an analysis of sequence similarity of multiple native archaeal tRNA sequences led to efficient, orthogonal suppressors of opal codons and the four-base codon, AGGA. Ultimately, it should be possible to use these additional orthogonal pairs to genetically incorporate multiple unnatural amino acids into proteins.

UI	MeSH Term	Description	Entries
D007935	Leucine-tRNA Ligase	An enzyme that activates leucine with its specific transfer RNA. EC 6.1.1.4.	Leucyl T RNA Synthetase,Leu-tRNA Ligase,Leucine-tRNA Synthetase,Leu tRNA Ligase,Leucine tRNA Ligase,Leucine tRNA Synthetase,Ligase, Leu-tRNA,Ligase, Leucine-tRNA,Synthetase, Leucine-tRNA
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009690	Nucleic Acid Conformation	The spatial arrangement of the atoms of a nucleic acid or polynucleotide that results in its characteristic 3-dimensional shape.	DNA Conformation,RNA Conformation,Conformation, DNA,Conformation, Nucleic Acid,Conformation, RNA,Conformations, DNA,Conformations, Nucleic Acid,Conformations, RNA,DNA Conformations,Nucleic Acid Conformations,RNA Conformations
D010957	Plasmids	Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS.	Episomes,Episome,Plasmid
D003062	Codon	A set of three nucleotides in a protein coding sequence that specifies individual amino acids or a termination signal (CODON, TERMINATOR). Most codons are universal, but some organisms do not produce the transfer RNAs (RNA, TRANSFER) complementary to all codons. These codons are referred to as unassigned codons (CODONS, NONSENSE).	Codon, Sense,Sense Codon,Codons,Codons, Sense,Sense Codons
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005796	Genes	A category of nucleic acid sequences that function as units of heredity and which code for the basic instructions for the development, reproduction, and maintenance of organisms.	Cistron,Gene,Genetic Materials,Cistrons,Genetic Material,Material, Genetic,Materials, Genetic
D006217	Halobacterium	A genus of HALOBACTERIACEAE whose growth requires a high concentration of salt. Binary fission is by constriction.
D000926	Anticodon	The sequential set of three nucleotides in TRANSFER RNA that interacts with its complement in MESSENGER RNA, the CODON, during translation in the ribosome.	Anticodons
D001483	Base Sequence	The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence.	DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA