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. (125)I-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 (125)I 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 (125)I is incorporatedseven nucleotides from the active center of RNAP, the yield of strand breaks is substantially lower than if (125)I 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 (125)I-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.

Radioprobing the conformation of DNA in a p53-DNA complex

“…Using the empirical dependence between these two parameters (Karamychev et al, 2000), we generated two sets of I-S distances, one for free DNA (white circles in Figure 4A), and another for the p53-DNA complex (black circles in Figure 4A). The two curves presented in Figure 5 (broken and solid grey lines) provide a reasonable basis for a semi-quantitative structural interpretation of the radioprobing results.…”

“…Therefore, the distance between 125 I incorporated in cytosine and the complementary guanine also remains the same in free DNA and in the complex. In this way, the relative changes in the intensity of breaks can be directly linked to the changes in DNA conformation, given that the frequency of breaks inversely correlates with the distance from the radioisotope to a given nucleotide – more specifically, to the iodine-sugar (I-S) distance (Karamychev et al, 1999, Panyutin and Neumann, 1997) (Karamychev et al, 2000). …”

“…The solid and broken grey lines represent the I-S distances for the p53-DNA complex and free DNA, respectively. The distances were calculated using the intensities of the single strand breaks in DNA (Figure 4A) and the empirical dependence between these two parameters (Karamychev et al, 2000). …”

“…Frequency of the single strand breaks in DNA produced by the Auger electrons emitted by decay of 125 I, inversely correlates with the distance from the radioisotope to the sugar ring in a given nucleotide (Karamychev et al, 1999, Panyutin and Neumann, 1997) (Karamychev et al, 2000). Therefore, frequencies of the 125 I-induced DNA breaks provide information on the DNA conformation.…”

“…The estimation of sensitivity of method indicates that, for example, the roll angles of 20-25° would be reliably detected (Karamychev et al, 1999). This method was also applied to characterize the spatial organization of the three DNA strands in the RecA filament (Malkov et al, 2000) and the DNA and RNA strands in the transcription elongation complex (Karamychev et al, 2000) (Karamychev et al, 2003). Here we employed radioprobing to determine structural changes in DNA RE upon binding to wt p53.…”