Site-specific mutagenesis on the 32-T and 39-T of Escherichia coli tRNA phe gene.
1989
Kim I.Y. | Lee S.Y.
There are three pseudouridine (psi) bases in the E. coli tRNA phe. In order to study the function of the pseudouridine bases in the tRNA phe, changes of bases tRNA phe gene to other bases were undertaken by the site-specific mutagenesis. Site-specific mutagenesis of T in the phe W gene, a tRNA phe gene of E. coli, corresponding to the base at the No. 32 position to C and also T corresponding to the base at the No. 39 position to C were performed using Kunkel's uracil-containing template method. Identification of mutants were undertaken by the DNA sequencing techniques of the mutated phe W genes and activities of the mutated phe W genes complementing to E. coli NP37 mutant (phe s** (ts)) using the recombinant plasmid containing the mutated genes. Neither NP37 harboring phe W gene mutated at No.32 position nor NP37 harboring phe W gene mutated at No. 39 position can be grown at non-permissive temperature. The result means that both mutated phe W genes can not complement to E. coli NP37, and that the pseudouridine bases are essential to the activity of the E. coli tRNA phe in vivo.
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