Fig. 6.

Role of nontemplate DNA in RDR2 engagement of RNAs in the context of RNA/DNA hybrids. (A) A nucleic acid configuration that mimics the situation when Pol IV arrests 15 nt into a downstream dsDNA region was prepared by annealing 39-nt RNA (R, red), 51-nt template DNA (T, green), and 28-nt nontemplate DNA strands (NT, green). RDR2 transcription was tested using R only (lane 1), R+T (lane 2), and R+T+NT combinations (lane 3), with transcripts labeled by incorporation of α-[³²P]ATP. (B) Test of nontemplate DNA/RNA overlap length. Same assay as in A, but varying the NT strand to have 10-, 5-, or 0-nt of sequence identity (overlap) with the RNA strand. (C) RNase I test of RNA displacement. 5′ end-labeled R strands in R only, R+T, or R+T+NT combinations were examined at four RNase I concentrations. (D) Graphic interpretation of the results in A–C.