Figure 3

Roles of TL amino acids in catalysis. (a). Reaction rates in saturating (1 mM) cGTP in EC^G1 and K_d [ncGTP] by wild-type (WT), M1238A, M1238V and M1238L are shown below the cartoons of the active centres of the corresponding enzymes, drawn in PyMol using PDB 2O5J and 'mutagenesis' function (colour code as in Figure 1). (b) Kinetics of pyrophosphorolysis by WT, R1239A and H1242A in the presence of 0.5 mM PP_i in EC^G1 (Additional File 1: Figure S2) with ³²P 5'-labelled RNA that was walked by two positions (G and A). (c) Kinetics of intrinsic transcript hydrolysis by WT (red squares), Q1235A (violet circles) and R1239A (green circles) RNA polymerase (RNAP) in EC^hydr (Additional File 1: Figure S2) with ³²P 5'-labelled RNA. The lines in the plot are the non-linear regression fits of the data. (d) pH dependences of the rates of 1 mM cGTP incorporation in EC^G1 at 20°C by WT, R1239A, H1242A and R1239A/H1239A RNAPs. Solid lines show fits of the data to a sigmoidal function, and pK_a values retrieved from these fits are shown above the plots. (e) Kinetics of 1 μM cGTP incorporation in EC^G1 by WT (red squares), R1239A (green circles) and R1239N (blue triangles). Solid lines show fits of the data to an exponential function.