Aspects of in Vivo and in Vitro Transcription in Yeast
Marczynski, Gregory Thadeus
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https://hdl.handle.net/2142/70570
Description
Title
Aspects of in Vivo and in Vitro Transcription in Yeast
Author(s)
Marczynski, Gregory Thadeus
Issue Date
1988
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Abstract
Yeast plasmids were employed to study the mechanisms of transcription by yeast RNA polymerase II and the yeast mitochondrial RNA polymerase both in vivo (in plasmid containing cells) and in vitro (in cell-free protein extracts). A complete in vivo transcription map of the yeast centromere plasmid YCp19 was determined, and the transcripts from the plasmid borne genes were quantitated. A plasmid position effect on gene expression was observed. Although YCp19 was maintained like a yeast minichromosome at approximately one plasmid per cell, several yeast genes overexpressed their RNA 3 to 5-fold above their chromosomal counterparts. The negative superhelical density ($-$d) was measured for yeast/E. coli shuttle plasmids isolated from yeast ($-$d = 0.02) and from E. coli ($-$d = 0.04). The superhelical density did not change when plasmids containing galactose inducible genes were induced to actively transcribe these genes. These results would favor models involving subtle DNA conformational changes to explain transcriptional activation.
Many attempts were made to reconstitute selective RNA polymerase II transcription initiation in vitro employing whole-cell protein extracts prepared from special genetically constructed yeast strains and employing supercoiled plasmid templates and the S1 nuclease protection assay. Although I failed to detect selective RNA polymerase II transcription initiation, I did observe selective transcription initiation by the yeast mitochondrial RNA polymerase from several nuclear genes. These mitochondrial RNA polymerase transcripts initiate from the RNA polymerase II "TATA" box promoter elements. Three of these genes are involved in galactose metabolism which is enigmatically influenced by the mitochondria. Also, reports in the literature suggest a physiological role for the mitochondrial RNA polymerase in the nucleus. A combination of genetic, biochemical, and molecular-biology techniques were employed to convincingly demonstrate that the mitochondrial RNA polymerase is not transcriptionally active in the nucleus.
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