Attachment of nascent RNA molecules to superhelical DNA.

Abstract

Comparisons are made of the attachment of nascent RNA molecules synthesized in vitro with Escherichia coli RNA polymerase on native bacteriophage PM2 DNA, a double-helical circular DNA with −110 superhelical twists, and on a derivative of this DNA with no superhelical twists. When RNA synthesis is terminated on either DNA by adding EDTA to chelate the Mg2+ ions, the nascent (incompletely polymerized) RNA chains remain bound to the DNA by bonds that involve fewer than 20 nucleotides of the RNA molecule. Differences between the two kinds of DNA are found when the complexes with nascent RNA are treated with sodium dedecyl sulfate or other agents that dissociate proteins (e.g. 3 m -CsCl; 5 m -urea; phenol). With such treatments, all RNA is released from the non-superhelical DNA, whereas only 40% of the RNA molecules are released from the superhelical DNA. The other 60% become bound even more tightly to that DNA, apparently by means of a hybrid helix that can involve up to 600 nucleotides of the bound RNA. It is suggested that the hybrid helix forms spontaneously on the superhelical DNA after denaturing the enzyme because the unwinding of the DNA helix needed to create the RNA-DNA helix relieves some of the strain that is known to exist in the superhelical DNA. Since the extensive hybrid helix does not exist before adding the detergent, the intact structure of the enzyme appears to be important to prevent its formation. Evidently RNA polymerase has a site that dissociates the polymerized portion of the RNA from the DNA as the enzyme moves along the template.