Transcription induces the formation of a stable RNA.DNA hybrid in the immunoglobulin alpha switch region.
We previously showed that the in vitro transcription of a negatively supercoiled plasmid containing the murine IgA switch region caused the formation of fewer supercoiled conformers of the plasmid due to the presence of a stable RNA.DNA hybrid. Here, we demonstrate that the RNA.DNA hybrid is approximately 140 nucleotides, and it forms regardless of the initial topological state of the transcription template. Transcription of the switch region in a relaxed closed circular plasmid generates positively supercoiled plasmid conformers that revert to their original relaxed state when treated with RNase H. Conformers that have incorporated a stable RNA transcript are also observed when nicked circular and linear plasmids containing the IgA switch sequences are transcribed with 32P-labeled nucleotide triphosphates. Once formed, the RNA.DNA hybrid is stable to both thermal and superhelical stress, tolerating temperatures in excess of 95 degrees C and restraining approximately 12 positive supercoils in the plasmid.
Reaban
M E
ME
Department of Microbiology, University of Alabama at Birmingham 35294.
Lebowitz
J
J
Griffin
J A
JA
D000818:Animals; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D015321:Gene Rearrangement; D007070:Immunoglobulin A / Q000235:genetics*; D007134:Immunoglobulin Switch Region / Q000235:genetics*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009692:Nucleic Acid Heteroduplexes; D009693:Nucleic Acid Hybridization; D009711:Nucleotides / Q000032:analysis; D014158:Transcription, Genetic; D014764:Viral Proteins
D007070:Immunoglobulin A; D009692:Nucleic Acid Heteroduplexes; D009711:Nucleotides; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases