Site-specific mutagenesis on cloned DNAs: generation of a mutant of Escherichia coli tyrosine suppressor tRNA in which the sequence G-T-T-C corresponding to the universal G-T-pseudouracil-C sequence of tRNAs is changed to G-A-T-C.

Abstract

We have cloned the Escherichia coli tyrosine-inserting amber suppressor tRNA gene into the recombinant single-strand phage M12mp3. By using the M13mp3SuIII+ recombinant phage DNA as template and an oligonucleotide bearing a mismatch as primer, we have synthesized in vitro an M13mp3SuIII heteroduplex DNA that has a single mismatch at a predetermined site in the tRNA gene. Transformation of E. coli with the heteroduplex DNA yielded M13 recombinant phages carrying a mutant suppressor tRNA gene in which the sequence G-T-T-C, corresponding to the universal G-T-pseudouracil-C sequence in E. coli tRNAs, is changed to G-A-T-C. The mutant DNA has been characterized by restriction mapping and by sequence analysis. In contrast to results with the wild-type suppressor tRNA gene, cells transformed with recombinant plasmids carrying the mutant tRNA gene are phenotypically Su-. Thus, the single nucleotide change introduced has inactivated the function of the tRNA gene. By using E. coli minicells for studying the expression in vivo of cloned tRNA genes, we have found that cells transformed with recombinant plasmids carrying the mutant tRNA gene contain very little, if any, mature mutant suppressor tRNA. In contrast, the predominant low molecular weight RNA in cells transformed with recombinant plasmids carrying the wild-type suppressor tRNA gene is the mature tyrosine suppressor tRNA. Thus, while our results imply an important role for the G-T-pseudouracil-C sequence common to all E. coli tRNAs, whether this sequence is essential for tRNA biosynthesis, tRNA stability in vivo, or tRNA function remains to be determined. The procedures used to generate the mutant should be of general application toward site-specific mutagenesis on cloned DNAs, including regions that possess high degrees of secondary structure. In addition, the frequency of mutants among the progeny is high enough to enable one to identify and isolate site-specific mutants on any cloned DNA without requiring phenotypic selection.