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Volume 271, Number 51, Issue of December 20, 1996 pp. 32707-32713
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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The Transcription Initiation Pathway of Sigma 54 Mutants That Bypass the Enhancer Protein Requirement
IMPLICATIONS FOR THE MECHANISM OF ACTIVATION

(Received for publication, May 16, 1996, and in revised form, August 15, 1996)

From the Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, California 90095

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.

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