MEASUREMENT OF THE FREQUENCY OF HISTONE DISPLACEMENT DURING THE IN-VITRO TRANSCRIPTION OF NUCLEOSOMES - RNA IS A COMPETITOR FOR THESE HISTONES

Transcription through tandemly arranged nucleosomes was studied to det ermine the frequency at which the nucleosomes would disrupt and cause displacement of the associated histones to a competitor DNA. In order to more effectively preserve topological effects, the template that wa s used in the in vitro transcription system was a large covalently, cl osed circular plasmid (8.9 kb). The plasmid contained two promoters fo r T7 RNA polymerase, each separated by 4.4 kb, and transcription was d one in the presence of topoisomerase I at physiological ionic strength . Nucleosome disruption was observed at an approximate frequency of 1 in 4 nucleosomes such that after several rounds of transcription on th e plasmid 80% of the nucleosomes were disrupted. Unexpectedly, all fou r histones were found associated with the RNA rather than the competit or DNA. The histones bound the competitor DNA only after removal of th e RNA by RNase A treatment. By analyzing the topological state of the competitor DNA, it was observed that the majority of the histones that were displaced from the RNA were able to re-form nucleosomes. Additio nal experiments were done to determine the reasons for the preferentia l binding of histones to the newly synthesized RNA. It was found that the large molecular weight RNA binds histones with an approximate 100- fold greater affinity relative to DNA when at physiological ionic stre ngth. Within the cell, this high affinity binding would be expected to require cellular mechanisms to regulate the interaction of RNA with h istones. The relatively high frequency of displacement of all four his tones during transcription is higher than what is observed in vivo and suggests that additional factors are needed to regulate this displace ment. These observations are discussed and compared with previous stud ies that have examined the process of transcription through nucleosome s.