The isolation of RNA homologous to the genetic control elements of the lactose operon

Abstract

A sequential DNA-RNA hybridization procedure is described whereby RNA homologous to a target DNA region 35 to 140 base pairs in length can be purified up to 6700-fold from a complex in vitro transcript to a homogeneity sufficient for sequence analysis. Requirements for the procedure include: (a) uniform transcription over the target DNA region in vitro; (b) specialized transducing phages which carry genetic deletions defining the target region on either side; and (c) specialized transducing phages which carry the target DNA in opposite orientations. These requirements have been met for the genetic control region (promoter, operator) of the lactose operon of Escherichia coli, to which the method was applied. The procedure is independent of the activity of the genetic control signals under study and can therefore be applied without modification to the study of point mutations introduced into the template.