Abstract
In vitro transcription, DNase I footprinting, and abortive initiation assays were used to characterize transcription using mutant forms of sigma 54 shown previously to bypass certain enhancer requirements in vitro. The holoenzymes containing these sigma mutants produce low levels of open complexes at both the glnAp2 and glnHp2 promoters. The open complexes are unusual in that they are destroyed by heparin. Enhancer protein and ATP convert them into a stable heparin-resistant state. The enhancer response occurs over a similar range of NtrC concentration as occurs with the wild-type holoenzyme, indicating that the activation determinants have been largely preserved within these mutants. One-round transcription assays show that the mutant holoenzymes can be driven to transcribe both promoters without NtrC. The unstable opening induced 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 enhancer complex overcomes an inhibitory effect of the sigma 54 leucine patch and unlocks the melting activity of the holoenzyme. Second, different sigma 54 determinants are used to drive stabilization of the open complexes, allowing the full transcription potential to be realized.
Highlights
In vitro transcription, DNase I footprinting, and abortive initiation assays were used to characterize transcription using mutant forms of sigma 54 shown previously to bypass certain enhancer requirements in vitro
It is necessary to further understand the relationship between the observed low levels of melting and the higher levels of in vitro transcription. We investigate these and other issues by expanding the range of assays used to study these two mutant forms of sigma 54. These include direct assays for the formation of certain critical intermediate complexes along the transcription pathway: DNase footprinting assays for closed and open complexes and an abortive initiation assay for the extent to which the mutant holoenzymes can make the first mRNA bond
The results suggested that the mutations allowed the holoenzyme to form open complexes with DNA, but that these might be unstable and in equilibrium with closed complexes
Summary
The combination of the enhancerbinding protein NtrC and ATP stimulates the low permanganate signal of the mutant holoenzymes, bringing it closer to the fully activated level The properties of these mutants should provide a substantial basis for learning how sigma 54 confers on polymerase the need for and ability to use enhancers and ATP. We investigate these and other issues by expanding the range of assays used to study these two mutant forms of sigma 54 These include direct assays for the formation of certain critical intermediate complexes along the transcription pathway: DNase footprinting assays for closed and open complexes and an abortive initiation assay for the extent to which the mutant holoenzymes can make the first mRNA bond. The results suggest an expanded model for the action of activators and for the role of the sigma 54 leucine patch in this process
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