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. Author manuscript; available in PMC: 2009 Jul 20.
Published in final edited form as: Anal Biochem. 2007 Apr 2;365(2):194–200. doi: 10.1016/j.ab.2007.03.039

Figure 3.

Figure 3

Stepwise elongation of the hairpin primer-template RNAs monitored by fluorescence polarization. The RNAs were pre-incubated with 3Dpol and ATP at 4°C for 30 minutes prior to being warmed up to room temperature for the FP assays. UTP was then added, triggering the addition of 2, 4, or 6 uracils depending on the length of the template sequence (Table 1) and stalling the 3Dpol-RNA complexes three nucleotides from the 5′ end of the template strand. The RNA was then fully elongated by the stepwise addition of the remaining NTPs. Note that regardless of the RNA length, only the incorporation of the penultimate nucleotide leads to a significant increase in the FP signal. We attribute this to an immobilization of the fluorescein probe on the protein surface as the 5′ end of the templating strand is translocated into the active site.