Fig 4.

Sensitivity of cis- and trans-priming to inhibition by PFA. (A) Priming reactions were performed in TMnNK using SUMO-MiniRT2 (1 pmol) alone (lanes 1 and 2) or together with increasing amounts of His-TP (1 pmol, lanes 3 and 6; 2 pmol, lanes 4 and 7; 4 pmol, lanes 5 and 8). The pyrophosphate analog PFA (at a 1 mM final concentration) was added to the priming reaction mixtures shown in lanes 2 and 6 to 8. The arrowhead denotes a degradation product from SUMO-MiniRT2 that apparently was able to serve as a primer in trans and most likely consisted of the SUMO tag plus the TP domain remaining attached (i.e., SUMO-TP). (B) trans-complementation priming reactions were performed in TMnNK using GST-RT (lanes 1 to 4) and His-TP (lanes 1 and 2) or His-TP-Y96F (lanes 3 and 4). PFA (at a 1 mM final concentration) was added to the reaction mixtures shown in lanes 3 and 4. Priming products were resolved by SDS-PAGE and detected by autoradiography. Priming signals were quantified using phosphorimaging and are represented at the graphs shown at the bottom, with those in the absence of PFA set at 100. The means and standard errors are shown. Statistical significance was calculated using the one-tailed, unpaired Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not statistically significant.