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. Author manuscript; available in PMC: 2022 Nov 15.
Published in final edited form as: Biochemistry. 2021 Oct 27;60(44):3323–3336. doi: 10.1021/acs.biochem.1c00528

Figure 4. Phosphoryl transfer in reverse.

Figure 4.

(A) Scaffold 2 and experimental overview of the pyrophosphorolysis assay. NT-DNA and T-DNA are noncomplementary in the region of the RNA-DNA hybrid. The RNA was 5′ end labeled in order to track the progress of the reaction. Apyrase was included to break down UTP released by the reaction and thus favor RNA cleavage. (B) Pyrophosphorolysis by WT and H1085L RNAPII. H1085L reduced the rate of U14 RNA decay by a factor of ~3 (unpaired two-sample t-test P=0.0037). See Figure S2 for an image of the full gel and kinetic fits of the data.