DNA and RNA Folds in Transcription Complex as Evidenced by Iodine-125 Radioprobing

Abstract

Folding of the DNA and RNA strands in an arrested T7 RNA polymerase (RNAP) transcription complex was studied by radioprobing, a novel method based on an analysis of the strand breaks produced by decay of the iodine-125 incorporated at the C5 position of cytosine. 125I-labeled cytosines were incorporated into transcripts at different positions relative to the site of the arrest. The intensities of the DNA breaks inversely correlate with the distances from the 125I decay site, so the radioprobing data provide information about the spatial RNA/DNA folding during transcription. We found that the yield of DNA strand breaks is significantly higher in the template than the non-template strand. This is consistent with local opening of the DNA duplex and formation of a hybrid between RNA and the template DNA strand. Our data demonstrate that the RNA-DNA hybrid has a nonuni form A-like structure. When the 125I is incorporatedseven nucleotides from the active center of RNAP, the yield of strand breaks is substantially lower than if 125I is positioned at the ends of the hybrid. Consequently, the DNA and RNA strands are located closer to each other at the ends of the hybrid and somehow separated in the middle. Surprisingly, the 125I-induced breaks were detected in both DNA strands upstream from the transcription “bubble” indicating that DNA and RNA are closely associated outside the RNAP cleft. Thus, radioprobing data imply that the RNA/DNA fold in the complex with T7 RNAP is more complicated than had been anticipated by the existing models. Based on the present data, we suggest a sterically feasible model explaining how formation of the long RNA-DNA hybrid can result in the initiation-to-elongation switch in the T7 transcription complex. According to this model, the topological linkage between the DNA and RNA strands provides the necessary stability for the elongation complex, while permitting movement of the polymerase along the DNA duplex.