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Nucleic Acids Research, 1986, Vol. 14, No. 14 5761-5776
© 1986

Articles

Site-directed mutagenesis of the binding site for ribosomal protein S8 within 16S ribosomal RNA from Escherichia coli

Richard J. Gregory and Robert A. Zimmermann

Department of Biochemistry, University of Massachusetts Amherst, MA 01003, USA

Received March 11, 1986. Revised June 16, 1986. Accepted June 16, 1986.

Twelve specific alterations have been introduced into the binding site for ribosomal protein S8 in Escherichla coli 16S rRNA. Appropriate rDNA segments were first cloned into bacteriophage M13 vectors and subjected to bisulfite and oligonucleotide-directed mutagenesis in vitro. Subsequently, the mutagenlzed sequences were placed within the rrnB operon of plasmid pN01301 and the mutant plasmids were used to transform E. coli recipients. The growth rates of cells containing the mutant plasmids were determined and compared with that of cells containing the wild-type plasmid. Only those mutations which occurred at highly conserved positions, or were expected to disrupt the secondary structure of the binding site, increased the doubling time appreciably. The most striking changes in growth rate resulted from mutations that altered a small internal loop within the S8 binding site. This structure is phylogenetlcally conserved in prokaryotic 16S rRNAs and may play a direct role In S8-16S rRNA recognition and interaction.


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