Relaxed Rotational and Scrunching Changes in P266L Mutant of T7 RNA Polymerase Reduce Short Abortive RNAs while Delaying Transition into Elongation
Figure 3
Promoter binding, opening, GTP binding and RNA turnover.
(A) TAMRA fluorophore labeled promoter DNA (20 nM) was titrated with increasing P266L T7 RNAP (0 to 100 nM) and increase in fluorescence anisotropy was fit to the quadratic equation with Kd of 3.6±1.1 nM. Similar to the WT T7 RNAP, the P266L mutant did not cause significant changes in the TAMRA fluorescence intensity (<10%) upon binding. Effect of the intensity changes on the fitting was insignificant and corrected the same way as reported previously [23]. Shown here are averaged values with standard deviations (error bars) from multiple independent measurements, from which Kd was fitted. (B) The increase in 2-AP fluorescence at −4 in the template strand upon addition of WT and P266L indicate slightly lower promoter opening with P266L. The errors are standard deviation from 10–15 measurements. (C) Initiating GTP binding was monitored from fluorescence increase in the 2-AP (−4 position) labeled promoter DNA bound to T7 RNAP titrated with increasing 3′dGTP. The P266L binds to the initiating NTPs (3′dGTP) ∼2 times weaker than WT T7 RNAP (175 μM versus 405 μM) and Hill coefficients are 1.3±0.02 and 1.7±0.03 for WT and P266L, respectively. The errors represent the fitting uncertainty. (D) A complex of WT or P266L T7 RNAP (2 µM) and promoter DNA (1 µM) was mixed with limited NTPs or NTP and 3′-deoxy NTP mixture for 2 min at 25°C to allow RNA synthesis of the indicated lengths. The amount of RNA shown in µM is representative of the number of turnovers at each walked position for WT (black) and P266L (grey) T7 RNAP.