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Mapping sigma 54-RNA polymerase interactions at the -24 consensus promoter element
Patricia C Burrows1, Konstantin Severinov, Akira Ishihama
1Department of Biological Sciences, Sir Alexander Fleming Building, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom.
The Journal of Biological Chemistry
|May 17, 2003
Summary
The sigma 54 RNA polymerase interacts with promoter DNA differently than sigma 70, with specific DNA-binding interactions changing during transcription initiation and elongation.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Sigma 54 (σ54) RNA polymerase (RNAP) is a distinct transcription factor that differs from sigma 70 (σ70)-type factors.
- σ54-RNAP binds promoters at conserved -24 and -12 elements and requires enhancer-binding activators for transcription initiation.
- The precise interface between σ54-RNAP and promoter DNA is not well understood, unlike the extensively studied σ70-promoter interactions.
Purpose of the Study:
- To physically map the σ54-RNAP interaction sites with promoter DNA using chemical cleavage methods.
- To investigate how these interactions change during the transition from closed to open promoter complexes and during transcription initiation.
- To compare σ54-RNAP promoter interactions with those of σ70-RNAP and identify unique features of σ54-dependent transcription.
Main Methods:
- Site-specific chemical modification of σ54 with cleavage reagents.
- Analysis of cleavage products to determine proximity between σ54 domains and promoter DNA elements (-24 and -12 regions).
- Investigating the role of core RNAP and DNA fork structures in promoter interactions.
Main Results:
- The conserved RpoN box motif of σ54 is positioned near the -24 promoter element, suggesting direct interaction.
- The spatial relationship between σ54-RNAP and the -24 element is stable during promoter complex formation and initiation but changes during elongation.
- Interactions with the -12 element change upon open complex formation, with DNA fork structures potentially substituting for core RNAP.
- The β-subunit flap domain mediates distinct interactions with promoter DNA in σ54- and σ70-dependent systems.
Conclusions:
- The RpoN box is a key σ54 DNA-binding motif interacting with the -24 promoter element.
- Dynamic changes in σ54-DNA interactions occur during transcription initiation and elongation, particularly at the -12 element.
- σ54-RNAP utilizes unique mechanisms, including potential DNA fork interactions, for promoter recognition and activation, differing from σ70-RNAP.