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. 2014 Jun 30;111(31):11311–11316. doi: 10.1073/pnas.1402531111

Fig. 2.

Fig. 2.

Functional assays of RNA/DNA duplex overhangs for sequence-defined pausing. (A) Activity assay of duplex substrates with various overhangs. Sequences of duplexes D4 and D6 appended either RNA 3′ or DNA 5′ overhangs (Left). The sequence-defined pausing site is denoted (white triangles). Activity assay of duplex substrates with various overhangs (Right). In vitro reconstituted TF telomerase or AMV RT were assayed with RNA/DNA duplexes and [α-32P]dGTP in the presence (+) or absence (−) of 0.5 mM dATP as denoted above the gel. A 32P end-labeled 7-mer oligonucleotide is included as a loading control (l.c.). The DNA primers TAGGGTTA (M1) and (TG)4TAGGGTTA (M7) extended by one [α-32P]dGTP with TdT were included as size markers. (B) TF telomerase is inactive with the D5 duplex that contains a DNA 5′ overhang. Activity assay of in vitro reconstituted TF telomerase and AMV RT with duplex substrate variants (D1–D6) appended with DNA 5′ overhangs. Substrates were extended by the enzyme with [α-32P]dGTP. A 32P end-labeled 7-mer oligonucleotide was included as a loading control (l.c.).