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Nucleic Acids Research, 1991, Vol. 19, No. 15 4259-4265
© 1991

MOLECULAR BIOLOGY

Mutations in 16S rRNA in Escherichia coli at methyl-modified sites: G966, C967, and G1207

David K. Jemiolo, Jacqueline S. Taurence and Sharon Giese

Biology Department Box 483, Vassar College, Poughkeepsie, NY 12601, USA

Received February 14, 1991. Revised July 3, 1991. Accepted July 3, 1991.

Mutations were constructed at three sites in 16S rRNA in E. coli by oligonucleotide-directed mutagenesis, and cloned into the rrnB operon on either pKK3535 or pNO2680. The mutated bases, G966, C967, and G1207, are located in the 3' major domain of 16S rRNA and are sites post-transcriptionally modified by methylation. We constructed a deletion mutation at C967 ({Delta}967) and three substitution mutations at each of the following sites: G966, C967, and G1207. By maxicell analysis, we found that all of the mutations were processed normally and incorporated into 30S subunits and 70S ribosomes. We found that {Delta}967 was a dominant lethal mutation while the substitution mutations at G966 and C967 had no effects on cell growth rate. The mutants C1207 and U1207 were shown to have dominant lethal phenotypes while A1207 had no effect on cell growth rate. These results help to establish the importance of methyl-modified regions to ribosome function.


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