THE TRANSCRIPTION INITIATION PATHWAY OF SIGMA-54 MUTANTS THAT BYPASS THE ENHANCER PROTEIN REQUIREMENT - IMPLICATIONS FOR THE MECHANISM OF ACTIVATION

In vitro transcription, DNase I footprinting, and abortive initiation assays were used to characterize transcription using mutant forms of s igma 54 shown previously to bypass certain enhancer requirements in vi tro. The holoenzymes containing these sigma mutants produce low levels of open complexes at both the glnAp2 and glnHp2 promoters. The open c omplexes are unusual in that they are destroyed by heparin. Enhancer p rotein and ATP convert them into a stable heparin-resistant state. The enhancer response occurs over a similar range of NtrC concentration a s occurs with the wildtype holoenzyme, indicating that the activation determinants have been largely preserved within these mutants. One-rou nd transcription assays show that the mutant holoenzymes can be driven to transcribe both promoters without NtrC. The unstable opening induc ed by these mutations apparently serves as a conduit that can shuttle templates into transcriptionally competent complexes. The results lead to a model in which activation occurs in two steps. First, the enhanc er complex overcomes an inhibitory effect of the sigma 54 leucine patc h and unlocks the melting activity of the holoenzyme. Second, differen t sigma 54 determinants are used to drive stabilization of the open co mplexes, allowing the full transcription potential to be realized.