R-loop formation in trans and its dependence on supercoiling of the target DNA. RNA containing an AGGAG repeat was produced from linearized pSK-(AGGAG)22, and the effects on a T7 promoterless plasmid containing an AGGAG repeat, pHC624-(AGGAG)22, were examined by agarose gel electrophoresis. Lane 1: pSK-(AGGAG)22 linearized with Xba I; lane 2: linearized pSK-(AGGAG)22 transcribed with T7 RNA polymerase; lane 3: supercoiled pHC624-(AGGAG)22, which lacked T7 promoter; lane 4: supercoiled pHC624-(AGGAG)22 incubated in transcription mixture with T7 RNA polymerase; lane 5: supercoiled pHC624-(AGGAG)22 and linearized pSK-(AGGAG)22 transcribed with T7 RNA polymerase; lane 6: supercoiled pHC624-(AGGAG)22 and pBluescript SK linearized with Xba I, transcribed with T7 RNA polymerase; lane 7: supercoiled pHC624; lane 8: supercoiled pHC624 and linearized pSK-(AGGAG)22 incubated in transcription mixture with T7 RNA polymerase; lane 9: pHC624-(AGGAG)22 relaxed with vaccinia topoisomerase I; lane 10: relaxed pHC624-(AGAAG)22 and linearized pSK-(AGGAG)22 transcribed with T7 RNA polymerase; lane 11: same as lane 5 and ethanol-precipitated; lane 12: same as lane 11 and treated with 90 units of E. coli RNase H (TaKaRa) in 40 mM Tris·HCl pH 7.7, 4 mM MgCl2, 1 mM DTT, 4% glycerol, and 0.003% bovine serum albumin at 37°C for 1 h.
