PMID:16669631

Citation	Walmacq, C, Rahmouni, AR and Boudvillain, M (2006) Testing the steric exclusion model for hexameric helicases: substrate features that alter RNA-DNA unwinding by the transcription termination factor Rho. Biochemistry 45:5885-95
Abstract	Typical hexameric helicases form ring-shaped structures involved in DNA replication. These enzymes have been proposed to melt forked DNA substrates by binding to, and pulling, one strand within their central channel, while the other strand is forced outside of the hexamer by steric exclusion and specific contacts with the outer ring surface. Transcription termination factor Rho also assembles into ring-shaped hexamers that are capable to use NTP-derived energy to unwind RNA and RNA-DNA helices. To delineate the potential relationship between helicase structural organization and unwinding mechanism, we have performed in vitro Rho helicase experiments with model substrates containing an RNA-DNA helix downstream from a Rho loading site. We show that a physical discontinuity (nick) inhibits RNA-DNA unwinding when present in the RNA but not in the DNA strand. Moreover, the presence of a 3'-overhanging DNA tail (Y-shaped substrate) does not affect initial Rho binding but can impair helicase activity. This inhibitory effect varies with the length of the tail, is independent of the identity (A or U) of the tail residues, and is also obtained when a biotin-streptavidin complex replaces the single-stranded DNA arm. However, it is readily relaxed upon moving the reporter RNA-DNA helix farther from the Rho loading site. The data indicate that the Rho helicase uses a steric exclusion mechanism whereby the initial formation of a productive Rho-transcript complex is a crucial rate-limiting event, while no specific interactions with the displaced strand are required. These results outline significant similarities as well as some differences in the mechanism of unwinding between Rho and other hexameric helicases which are discussed in relation with the biological function of the Rho helicase.
Links	PubMed Online version:10.1021/bi0600648
Keywords	DNA/chemistry; DNA Helicases/chemistry; DNA Helicases/metabolism; Electrophoresis, Polyacrylamide Gel; Models, Molecular; RNA/chemistry; RNA Helicases/chemistry; RNA Helicases/metabolism; Substrate Specificity; Transcription, Genetic; rho GTP-Binding Proteins/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
ECOLI:RHO	involved_in	GO:0006351: transcription, DNA-templated	ECO:0000314: direct assay evidence used in manual assertion		P		Seeded From UniProt	complete

References

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