This proposal is aimed at constructing synthetic tRNA nonsense suppressor genes, which will direct the in vivo synthesis of a series of nonsense (in most cases UAG) condon. The goal is to derive a set of suppressors capable of inserting each of the 20 amino acids at a UAG site. This would greatly aid systematic studies of amino acid substitutions in proteins, since UAG mutations can be introduced by in vivo and in vitro methods at any position in a protein. The information gained from virtual unlimited amino acid substitutions would facilitate protein engineering experiments. The synthetic genes are put together from a series of oligonucleotides and introduced into E. coli cells by a plasmid vector. Additional modifications are introduced by site-directed mutagenesis, by random mutagenesis followed by selection, and by constructing hybrids of different tRNA molecules. The amino acid inserted by each suppressor is determined by protein sequencing in a system that employs an amber mutation in the gene encoding dihydrofolate reductase. Mutants will be sought which improve the specificity of certain suppressors by limiting the mischarging by non-cognate synthetases, such as glutamine tRNA synthetase. Different vector systems will be used to carry the suppressor genes to increase the general utility of the suppressors to different investigators. The goal of this proposal is to facilitate the engineering of specific amino acid replacements in proteins. In principle, new amino acids can be inserted at any site in the product of a cloned gene by a chemical procedure known as site-directed mutagenesis. However, this procedure becomes laborious when large numbers of specific replacements are needed. In collaboration with Dr. John Abelson at the California Institute of Technology, Dr. Miller intends to circumvent this problem by constructing transfer RNA genes that will perform the substitutions in vivo. The transfer RNA genes, known as suppressors, will be constructed so as to allow insertion of any one of the 20 different amino acids whenever the nonsense codon UAG occurs. Since this codon can be created at any location in a cloned gene, virtually all amino acid replacements will be possible. This collection of suppressor genes will be extremely useful to scientists studying the relationships between the structure and function of proteins. Important information on the functioning of transfer RNA molecules will also be obtained.
