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. / Graduation date: 1993
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/36227 |
| Date | 14 January 1993 |
| Creators | Georgel, Philippe, 1961- |
| Contributors | van Holde, Ken |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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