Multiple in vivo roles for the -12-region elements of sigma 54 promoters.

Alignment of sigma 54-dependent promoters indicates conservation of two sequence elements. Six nucleotides in the downstream -12 element were mutated individually to each nonconsensus nucleotide. mRNA levels were measured in vivo for each promoter under strongly activating conditions. The results showed that the consensus sequence was not the strongest promoter. Instead, the -12 consensus element consists of two subregions that behave differently when mutated. Single changes in the upstream TTT consensus subregion can lead to increases in transcription, whereas single changes in the downstream GC(A/T) can lead to decreases in transcription. Selected double mutations with changes in both subregions were constructed and studied in vivo. No double mutation increased promoter strength, and some decreased it. Mutant promoters were also assayed under nonactivating conditions in vivo. No mRNA was detected in 23 of the 24 promoters tested. However, one double mutant showed substantial levels of transcript, indicating that the -12 sequence was capable of specifying basal transcription under nonactivating conditions. Overall, the results show that the -12 region has multiple roles in transcription in vivo, including modulating both basal and induced RNA levels.