The ATP-dependent remodeler RSC transfers histone dimers and octamers through the rapid formation of an unstable encounter intermediate.
RSC, an essential chromatin remodeling complex in budding yeast, is involved in a variety of biological processes including transcription, recombination, repair, and replication. How RSC participates in such diverse processes is not fully understood. In vitro, RSC uses ATP to carry out several seemingly distinct reactions: it repositions nucleosomes, transfers H2A/H2B dimers between nucleosomes, and transfers histone octamers between pieces of DNA. This raises the intriguing mechanistic question of how this molecular machine can use a single ATPase subunit to create these varied products. Here, we use a FRET-based approach to kinetically order the products of the RSC reaction. Surprisingly, transfer of H2A/H2B dimers and histone octamers is initiated on a time scale of seconds when assayed by FRET, but formation of stable nucleosomal products occurs on a time scale of minutes when assayed by native gel. These results suggest a model in which RSC action rapidly generates an unstable encounter intermediate that contains the two exchange substrates in close proximity. This intermediate then collapses more slowly to form the stable transfer products seen on native gels. The rapid, biologically relevant time scale on which the transfer products are generated implies that such products can play key roles in vivo.
Rowe
Claire E
CE
Department of Biochemistry and Biophysics, University of California, San Francisco, California 94158, USA.
Narlikar
Geeta J
GJ
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002843:Chromatin / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006657:Histones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D009687:Nuclear Proteins / Q000378:metabolism; D009707:Nucleosomes / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification / Q000378:metabolism*; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic; D014981:Xenopus
D002843:Chromatin; D004268:DNA-Binding Proteins; D006657:Histones; D009687:Nuclear Proteins; D009707:Nucleosomes; C103337:RSC complex, S cerevisiae; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D000255:Adenosine Triphosphate