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. Author manuscript; available in PMC: 2012 Jan 21.
Published in final edited form as: J Mol Biol. 2010 Oct 28;405(3):666–678. doi: 10.1016/j.jmb.2010.10.020

Fig. 6. UMP incorporation during RNA synthesis measured by the radiometric assay.

Fig. 6

(A) The polyacrylamide sequencing gels show 10-mer RNA elongated to 11-13 mer RNAs with increasing reaction times. Lanes 0-12 represent reaction times from 0, 2, 4, 6, 8, 10, 30, 50, 100, 500 msec, and 1, 5, 10 s at 10 μM (right side) or 100 μM (left side) UTP. (B) The kinetics of 11-13 mer RNA formation at UTP concentrations of 10μM (filled circles) and 100 μM (filled triangles) were fit to a single exponential equation (Eq.1) with kobs = 24 s-1 and 113 s-1, respectively. (C) UTP concentration dependence of kobs for correct UMP incorporation fit to the hyperbolic function (Eq. 2) with kpol = 251 ± 23 s-1 and Kd = 133 ± 20 μM. (D) UTP concentration dependence of the kobs for rU:dA misincorporation fits to a line with slope = 0.001 μM-1.s-1. (E) UTP concentration dependence of the amplitudes of correct (filled circle) and incorrect (open circle) UMP incorporation. A net dissociation constant for UMP misincorporation (Kd,net-mis= 263 μM) was derived from the hyperbolic fitting.