The effect of promoter strength, supercoiling and secondary structure on mutation rates in Escherichia coli.

Four mutations resulting in opal stop codons were individually engineered into a plasmid-borne chloramphenicol-resistance (cat) gene driven by the lac promoter. These four mutations were located at different sites in secondary structures. The mutations were analysed with the computer program mfg, which predicted their relative reversion frequencies. Reversion frequencies determined experimentally correlated with the mutability of the bases as predicted by mfg. To examine the effect of increased transcription on reversion frequencies, the lac promoter was replaced with the stronger tac promoter, which resulted in 12- to 30-fold increases in reversion rates. The effect of increased and decreased supercoiling was also investigated. The cat mutants had higher reversion rates in a topA mutant strain with increased negative supercoiling compared with wild-type levels, and the cat reversion rates were lower in a topA gyrB mutant strain with decreased negative supercoiling, as predicted.