An efficiently mutagenizable recombinant plasmid for in vitro transcription of the Escherichia coli 16 S RNA gene.

The portion of the rrnB operon coding for 16 S RNA was modified to permit efficient in vitro transcription by T7 RNA polymerase of full-length, correctly terminated, biologically active 16 S RNA (W. Krzyzosiak et al., 1987, Biochemistry 26, 2353-2364). The 5'-end of the gene was fused to the class III T7 promoter and the 3'-end was modified so that cleavage with MstII would generate correctly terminated RNA upon runoff transcription. The modified gene was placed in pUC19 by a four-way ligation reaction involving linearized pUC19, a 1490-bp fragment of 16 S rDNA, and two synthetic oligodeoxynucleotides. Because of the cohesive end design, phosphorylation of the synthetic oligomers was not necessary. Single and tandem cassette insertions were used to generate single base changes in the C-1400 region of 16 S RNA. Three examples are described. This method is generally applicable to the 16 S RNA molecule as suitable singlecleavage restriction sites allow all regions to be mutated by this approach.