Photochemical crosslinking of transcription complexes with psoralen. I. Covalent attachment of in vitro SV4O nascent RNA to its double-stranded DNA template

Abstract

14c-labeled SV40 DNA has been transcribed with E. coli RNA polymerase using ³H-labeled ribonucleotide triphosphates as precursors. The resulting transcription complexes were then photochemically crosslinked with the psoralen derivative, 4′ -aminomethyl-4,5′ ,8-trimethylpsoralen (AMT), at 37°C and analyzed in SDS-sucrose gradients. It was found that the photochemical crosslinking procedure caused the nascent RNA chains to co-sediment with their double-stranded (helical) Sv40 templates in the denaturing sucrose gradient. This result and several control experiments suggest that covalent linkages have formed between nascent RNA and helical DNA after the photochemical reaction. The crosslinking phenomenon was observed to be independent of the superhelical state of the DNA used as the template. Prior addition of EDTA to stop the transcription is not requiréd for successful crosslinkage.