Role of the σ54 Activator Interacting Domain in Bacterial Transcription Initiation
Bacterial sigma factors are subunits of RNA polymerase that direct the holoenzyme to specific sets of promoters in the genome and are a central element of regulating transcription. Most polymerase holoenzymes open the promoter and initiate transcription rapidly after binding. However, polymerase containing the members of the σ54 family must be acted on by a transcriptional activator before DNA opening and initiation occur. A key domain in these transcriptional activators forms a hexameric AAA + ATPase that acts through conformational changes brought on by ATP hydrolysis. Contacts between the transcriptional activator and σ54 are primarily made through an N-terminal σ54 activator interacting domain (AID). To better understand this mechanism of bacterial transcription initiation, we characterized the σ54 AID by NMR spectroscopy and other biophysical methods and show that it is an intrinsically disordered domain in σ54 alone. In this paper, we identified a minimal construct of the Aquifex aeolicus σ54 AID that consists of two predicted helices and retains native-like binding affinity for the transcriptional activator NtrC1. Using the NtrC1 ATPase domain, bound with the non-hydrolyzable ATP analog ADP-beryllium fluoride, we studied the NtrC1–σ54 AID complex using NMR spectroscopy. We show that the σ54 AID becomes structured after associating with the core loops of the transcriptional activators in their ATP state and that the primary site of the interaction is the first predicted helix. Finally, understanding this complex, formed as the first step toward initiation, will help unravel the mechanism of σ54 bacterial transcription initiation.
- Research Organization:
- Univ. of California, Berkeley, CA (United States)
- Sponsoring Organization:
- USDOE; National Inst. of Health (NIH) (United States); National Science Foundation (NSF)
- DOE Contract Number:
- AC02-05CH11231; GM 62163; RR15756; GM68933; BBS 87-20134; BBS 01-19304
- OSTI ID:
- 1393088
- Journal Information:
- Journal of Molecular Biology, Vol. 428, Issue 23; ISSN 0022-2836
- Publisher:
- Elsevier
- Country of Publication:
- United States
- Language:
- English
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