TRANSCRIPTION INDUCES THE FORMATION OF A STABLE RNA-CENTER-DOT-DNA HYBRID IN THE IMMUNOGLOBULIN-ALPHA SWITCH REGION

We previously showed that the in vitro transcription of a negatively s upercoiled plasmid containing the murine IgA switch region caused the formation of fewer supercoiled conformers of the plasmid due to the pr esence of a stable RNA.DNA hybrid. Here, we demonstrate that the RNA.D NA hybrid is approximately 140 nucleotides, and it forms regardless of the initial topological state of the transcription template. Transcri ption of the switch region in a relaxed closed circular plasmid genera tes positively supercoiled plasmid conformers that revert to their ori ginal relaxed state when treated with RNase H. conformers that have in corporated a stable RNA transcript are also observed when nicked circu lar and linear plasmids containing the IgA switch sequences are transc ribed with P-32-labeled nucleotide triphosphates. Once formed, the RNA .DNA hybrid is stable to both thermal and superhelical stress, tolerat ing temperatures in excess of 95 degrees C and restraining approximate ly 12 positive supercoils in the plasmid.