Coupled-enzymatic assays for the rate and mechanism of DNA site exposure in a nucleosome.
The packaging of DNA in nucleosomes presents obstacles to the action of gene regulatory proteins and polymerases on their natural chromatin substrates. We recently reported that nucleosomes exist in a conformational equilibrium, transiently exposing stretches of their DNA off the histone surface. Such "site exposure" processes potentially provide the needed access of proteins to DNA in chromatin. However, the experiments that reveal site exposure are carried out on timescales of tens of minutes to hours. The actual rates of site exposure are not known. Here we use T7 RNA polymerase and exonuclease III as probes to obtain a more relevant lower bound on the rate of nucleosomal site exposure. We find that the organization of DNA into nucleosomes detectably slows the elongation rate of the polymerase, but that full-length elongation, which requires access to all of the DNA, occurs on the seconds timescale. Independent experiments with exonuclease III, which probes the outermost DNA segments only, similarly show that site exposure in these regions occurs on a timescale of seconds or faster. We conclude that site exposure is sufficiently rapid that it may play a role in the initial binding of regulatory proteins to nucleosomal target sites. These rapid rates argue against a nucleosome sliding model for the mechanism of site exposure. Surprisingly, the measured rates may be too slow to account for the known rates of polymerase elongation in vivo. Mechanisms by which polymerase progression through nucleosomes might be catalyzed are discussed.
Protacio
R U
RU
Department of Biochemistry Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208-3500, USA.
Polach
K J
KJ
Widom
J
J
D004247:DNA / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases; D005090:Exodeoxyribonucleases; D007700:Kinetics; D009707:Nucleosomes / Q000201:enzymology / Q000378:metabolism*; D013698:Templates, Genetic; D013997:Time Factors; D014158:Transcription, Genetic / Q000502:physiology*; D014764:Viral Proteins
D009707:Nucleosomes; D014764:Viral Proteins; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III