Fig. 4.

Comparative efficiency of Ap₄A, Gp₄A, Cp₄A, and Up₄A incorporation by E. coli RNA polymerase. (A) Absolute efficiency of Np₄A incorporation. RNA was synthesized in vitro by E. coli RNA polymerase holoenzyme in the presence of a mixture of each dinucleoside tetraphosphate and ATP. The promoter sequence at positions −3, −2, and −1 of the coding strand was AAN, where N = A, G, C, or T. In each case, the product ratio (capped transcripts/uncapped transcripts) was determined by boronate gel electrophoresis and normalized to the substrate ratio (Np₄A/ATP = 0.50). Each value corresponds to the mean of three technical replicates. Error bars represent 1 SD. P ≤ 0.003 for each AAPu vs. AAPy comparison involving the same Np₄A, where Pu = A or G and Py = C or T. (B) Relative efficiency of Np₄A incorporation. For each promoter in A, the efficiencies of Ap₄A, Gp₄A, Cp₄A, and Up₄A incorporation were normalized to the mean efficiency of all four with the same promoter. The relative efficiencies calculated in this manner can range from 0 to 4, with a relative efficiency of 1 being average for that promoter. Each value was calculated by using data from three technical replicates for each of the four Np₄As and a particular promoter. Error bars correspond to a confidence level of 68.3%, equivalent to 1 SD. ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05. ns, not significant (P > 0.05).