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. 2010 Nov 4;39(5):1749–1762. doi: 10.1093/nar/gkq935

Figure 6.

Figure 6.

Mechanism of RNA:DNA hybrid formation during in vitro transcription. (A) Schematic of the two possible mechanisms for R-loop formation. By the thread-back model, the nascent transcript (depicted in light blue) that has been ejected from the RNA polymerase re-anneals with the complementary, free DNA template strand (depicted in red) following the progression of the RNA polymerase (light blue, oval). When transcription occurs in the presence of RNAse A (dark blue) the nascent transcript is degraded when it is ejected from the RNA polymerase hence cannot form the hybrid. By the extended-hybrid model, the nascent transcript remains bound to the template DNA and resists becoming ejected from the RNA polymerase. When transcribed in the presence of RNase A, as the nascent transcript is protected by being bound to the template DNA it is not degraded hence hybrid formation is not ablated. (B) FRAXA plasmid (CGG)39 · (CCG)39 transcribed in either direction in the absence (−) or presence (+) of RNase A during the transcription reaction. All transcription reactions were subjected to further RNase A or A + H treatment to analyze hybrid formation. (C) Same experiment as in (B) performed with SCA1 plasmid containing a (CAG)74 · (CTG)74 repeat tract.