Effect of in vitro transcription on cruciform stability

We have investigated the effect of in vitro transcription on cruciform stability. Replicative form DNA of phiX174 strain ins6240, containing a 48 bp synthetic palindrome in the J-F intercistronic region, was supercoiled in vitro to mean negative superhelical densities (sigma) ranging from 0 to 0.15. The presence of cruciforms was probed by limited digestion with the single-strand specific nuclease Bal31. The 48 bp palindrome was extruded at a mean sigma = -0.05, but only after heating the DNA. An in vitro transcription reaction with E. coli RNA polymerase and [alpha-32P]UTP gave identical transcripts with heated or unheated template DNA. The synthetic cruciform was stable upon binding of the RNA polymerase to the template, but it was destabilized upon movement of the transcription complex along the template. Transcription of unheated templates did not result in cruciform formation. We propose that cruciform structures in supercoiled template DNAs present no hindrance to RNA polymerase, and thus have no detectable effect on transcription elongation in vitro.