Graduate Thesis Or Dissertation
 

Effects of nucleosomes on transcription by polymerase I in a reconstituted system

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/h128nj89w

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  • The aim of this study was to gain more information about the interactions between DNA and the histone octamer during the process of transcription. This work used a pUC8 plasmid derivative that contained the core promoter region of the RNA polymerase I of Acanthamoeba castellanii, placed upstream of four repeats of the 5S rDNA nucleosome positioning sequence from the sea urchin, Lytechinus variegatus. The plasmid was reconstituted into chromatin via addition of chicken erythrocyte histone octamers, using polyglutamic acid as a nucleosome assembly factor. The positioning of nucleosomes on the insert was monitored by restriction enzyme digestion. Proper nucleosome positioning was shown to be dependent on the presence of preassembled transcription complexes on the promoter region. The absence of preformed transcription complexes on the promoter region prior to nucleosome reconstitution perturbed the distribution of histone octamers on the repeats of the 5S rDNA. This "mispositioning" effect was related to the location of the RNA polymerase I promoter region upstream of the four repeats of the 5S rDNA fragment. Band shift assays in polyacrylamide gel electrophoresis were used to determine the relative efficiency of nucleosome formation on the promoter-containing fragment, on 5S rDNA and finally on nucleosome core particle DNA. The results indicate that the promoter fragment forms a nucleoprotein complex at lower concentration of histone than the 5S positioning sequence. This complex may not be a nucleosomal structure. The reconstituted plasmid was then used to investigate the transcriptional elongation by RNA polymerase I using the chromatin-like template containing positioned nucleosomes as compared to transcription on improperly positioned nucleosomes and on free DNA. The efficiency of transcription was related to the proper positioning of nucleosomes with regard to the tandemly repeated 208-bp 5S rDNA. The presence of phased nucleosomes in the path of the transcription complex seemed not to inhibit nor to significantly slow down the elongation as compared to free DNA. Furthermore, nucleosome positioning, as assayed by restriction endonuclease digestion, did not change after passage of the polymerase I transcription complex.
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