Molecular cloning of the gene for the RNA-processing enzyme RNase III of Escherichia coli

Abstract

A ColE1 plasmid from the Clarke and Carbon collection [Clarke, L. & Carbon, J. (1976) Cell 9, 91-99] that contains a 14.4-kilobase Escherichia coli DNA insert complements the rnc-105 mutation, which destroys the activity of the RNA-processing enzyme RNase III. This insert and smaller restriction endonuclease fragments derived from it were cloned into the plasmid pBR329. A number of these recombinant plasmids complemented the rnc-105 mutation in a recA genetic background. The smallest cloned fragment that compensated for the rnc-105 mutation was 1.3 kilobase in size. This fragment led to the synthesis of two polypeptides. One of these polypeptides was 25,300 daltons and corresponded in size to the subunit of RNase III. Fragments cloned in opposite orientations led to synthesis of RNase III, indicating that the cloned fragments contained an endogenous promoter. Extracts of an rnc+ E. coli strain containing an rnc+ plasmid had at least 10 times more RNase III activity than did an analogous strain containing the pBR329 plasmid.