کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
5533359 | 1402118 | 2016 | 17 صفحه PDF | دانلود رایگان |
- In free Ï54, the AID (residues 1-50) is intrinsically disordered.
- The AID becomes ordered upon core polymerase binding.
- The AID alone binds transcriptional activators in their ATP state.
- The AID binds the activator with native-like affinity.
- Ï54 residues 16-25 are the major contact region to the ATPase.
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 Ï54 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 AAAÂ + ATPase that acts through conformational changes brought on by ATP hydrolysis. Contacts between the transcriptional activator and Ï54 are primarily made through an N-terminal Ï54 activator interacting domain (AID). To better understand this mechanism of bacterial transcription initiation, we characterized the Ï54 AID by NMR spectroscopy and other biophysical methods and show that it is an intrinsically disordered domain in Ï54 alone. We identified a minimal construct of the Aquifex aeolicus Ï54 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-Ï54 AID complex using NMR spectroscopy. We show that the Ï54 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. Understanding this complex, formed as the first step toward initiation, will help unravel the mechanism of Ï54 bacterial transcription initiation.
Graphical Abstract112
Journal: Journal of Molecular Biology - Volume 428, Issue 23, 20 November 2016, Pages 4669-4685