Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10750380 | Biochemical and Biophysical Research Communications | 2016 | 23 Pages |
Abstract
Promoter recognition by bacterial RNA polymerase (RNAP) is a multi-step process involving multiple protein-DNA interactions and several structural and kinetic intermediates which remain only partially characterized. We used single-stranded DNA aptamers containing specific promoter motifs to probe the interactions of the Thermus aquaticus RNAP ÏA subunit with the â10 promoter element in the absence of other parts of the promoter complex. The aptamer binding decreased intrinsic fluorescence of the Ï subunit, likely as a result of interactions between the â10 element and conserved tryptophan residues of the Ï DNA-binding region 2. By monitoring these changes, we demonstrated that DNA binding proceeds through a single rate-limiting step resulting in formation of very stable complexes. Deletion of the N-terminal domain of the ÏA subunit increased the rate of aptamer binding while replacement of this domain with an unrelated N-terminal region 1.1 from the Escherichia coli Ï70 subunit restored the original kinetics of Ï-aptamer interactions. The results demonstrate that the key step in promoter recognition can be modelled in a simple Ï-aptamer system and reveal that highly divergent N-terminal domains similarly modulate the DNA-binding properties of the Ï subunit. The aptamers efficiently suppressed promoter-dependent transcription initiation by the holoenzyme of RNA polymerase, suggesting that they may be used for development of novel transcription inhibitors.
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Authors
Nataliya Miropolskaya, Andrey Kulbachinskiy,