Transcribed Guanine-rich RNA Aggregates on Template DNA and Changes Its Conformation

Abstract

The direct visualization of transcribed RNA-dependent DNA structural change by atomic force microscopy revealed that guanine-rich RNA acts as a molecular switch for changing DNA conformation and the changed DNA structure is stably maintained, suggesting the possibility that DNA can memorize past transcription action in terms of shape.DNA, encoding genetic information, is a flexible material that can change its conformation considerably. Although the conformational change of DNA is important in many biological reactions, 1 its precise mechanism has been insufficiently analyzed. Previously, we suggested that guanine-rich RNA (Grich RNA) transcripts aggregate on template DNA, change the DNA conformation and accelerate several biological processes.2,3 For example, immunoglobulin (Ig) switch-region RNA aggregates on template DNA after transcription, generates a large RNA and DNA complex (RNA/DNA complex), and relaxes the template DNA, which may be essential processes for the DNA recombination of Ig genes (class switch recombination) during B lymphocyte differentiation.2 The precise structure of the RNA/DNA complex, however, has not been sufficiently clarified. Here, using two ribonucleases (RNase A and RNase H), we degraded aggregated RNA in a stepwise manner and examined the conformational change of template DNA by agarose gel electrophoresis and tapping-mode atomic force microscopy (AFM).The plasmid DNA pGD231 contains the Ig switch-region DNA fragment. Using this plasmid, we generated G-rich RNA using T7 RNA polymerase in vitro (in vitro transcription) as reported previously.2 The transcribed samples were initially treated with RNase A and then with RNase H. An aliquot of each sample was analyzed by agarose gel electrophoresis or AFM. Briefly, the samples for agarose gel analysis were run on a 1.5% gel in TAE buffer (40 mM Tris-Acetate, 1 mM EDTA) at 5 V cm ¹1