Figure 2.

The template 5′-flanking region length contributes to activity and defining the template 5′-boundary. (A) Activity assay of hTRmin 41–43 indel mutants reconstituted in RRL. Diagrams of template 5′-flanking region length changes are shown with template in pink, AGA59–61 in green, UUU41–43 in yellow, and added sequence in darker gray. The assays were carried out using primer T₂₁-GTTAGG in presence of all 4 dNTPs or omitting dCTP. (B) Activity assay of 59–61 and 41–43 combination mutants reconstituted in RRL in presence of all 4 dNTPs or omitting dCTP. Blue dots indicate products from template 5′ boundary bypass observed only in the presence of dCTP. Values of rRAP and rAct were calculated as described in Figure 1. Similar results were obtained from 2 independent experimental replicates. (C) Model for activity and RAP defects induced by length changes in the template 5′- and 3′-flanking single-stranded RNA regions and P2a.1 stem disruption. Schematic shows template position post-translocation with TEN domain of TERT near the 3′ end of template-product hybrid.