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Elucidating the mechanism of DNA-dependent ATP hydrolysis mediated by DNA-dependent ATPase A, a member of the SWI2/SNF2 protein family.

ABSTRACT: The active DNA-dependent ATPase A domain (ADAAD), a member of the SWI2/SNF2 family, has been shown to bind DNA in a structure-specific manner, recognizing DNA molecules possessing double-stranded to single-stranded transition regions leading to ATP hydrolysis. Extending these studies we have delineated the structural requirements of the DNA effector for ADAAD and have shown that the single-stranded and double-stranded regions both contribute to binding affinity while the double-stranded region additionally plays a role in determining the rate of ATP hydrolysis. We have also investigated the mechanism of interaction of DNA and ATP with ADAAD and shown that each can interact independently with ADAAD in the absence of the other. Furthermore, the protein can bind to dsDNA as well as ssDNA molecules. However, the conformation change induced by the ssDNA is different from the conformational change induced by stem-loop DNA (slDNA), thereby providing an explanation for the observed ATP hydrolysis only in the presence of the double-stranded:single-stranded transition (i.e. slDNA).

SUBMITTER: Nongkhlaw M

PROVIDER: S-EPMC2691824 | biostudies-other | 2009 Jun

REPOSITORIES: biostudies-other

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Elucidating the mechanism of DNA-dependent ATP hydrolysis mediated by DNA-dependent ATPase A, a member of the SWI2/SNF2 protein family.

Nongkhlaw Macmillan M Dutta Popy P Hockensmith Joel W JW Komath Sneha Sudha SS Muthuswami Rohini R

Nucleic acids research 20090326 10

The active DNA-dependent ATPase A domain (ADAAD), a member of the SWI2/SNF2 family, has been shown to bind DNA in a structure-specific manner, recognizing DNA molecules possessing double-stranded to single-stranded transition regions leading to ATP hydrolysis. Extending these studies we have delineated the structural requirements of the DNA effector for ADAAD and have shown that the single-stranded and double-stranded regions both contribute to binding affinity while the double-stranded region a ...[more]

PMID: 19324887

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Project description:Free-living bacteria have regulatory systems that can quickly reprogram gene transcription in response to changes in cellular environment. The RapA ATPase, a prokaryotic homolog of the eukaryote Swi2/Snf2 chromatin remodeling complex, may facilitate such reprogramming, but the mechanisms by which it does so is unclear. We used multi-wavelength single-molecule fluorescence microscopy in vitro to examine RapA function in the E. coli transcription cycle. In our experiments, RapA at < 5 nM concentration did not appear to alter transcription initiation, elongation, or intrinsic termination. Instead, we directly observed a single RapA molecule bind specifically to the kinetically stable post-termination complex (PTC) -- consisting of core RNA polymerase (RNAP) bound to dsDNA -- and efficiently remove RNAP from DNA within seconds in an ATP-hydrolysis-dependent reaction. Kinetic analysis elucidates the process through which RapA locates the PTC and the key mechanistic intermediates that bind and hydrolyze ATP. This study defines how RapA participates in the transcription cycle between termination and initiation and suggests that RapA helps set the balance between global RNAP recycling and local transcription re-initiation in proteobacterial genomes.SignificanceRNA synthesis is an essential conduit of genetic information in all organisms. After transcribing an RNA, the bacterial RNA polymerase (RNAP) must be reused to make subsequent RNAs, but the steps that enable RNAP reuse are unclear. We directly observed the dynamics of individual molecules of fluorescently labeled RNAP and the enzyme RapA as they colocalized with DNA during and after RNA synthesis. Our studies show that RapA uses ATP hydrolysis to remove RNAP from DNA after the RNA is released from RNAP and reveal essential features of the mechanism by which this removal occurs. These studies fill in key missing pieces in our current understanding of the events that occur after RNA is released and that enable RNAP reuse.

Dataset Information

Elucidating the mechanism of DNA-dependent ATP hydrolysis mediated by DNA-dependent ATPase A, a member of the SWI2/SNF2 protein family.

Publications

Elucidating the mechanism of DNA-dependent ATP hydrolysis mediated by DNA-dependent ATPase A, a member of the SWI2/SNF2 protein family.

Similar Datasets

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