A study of the regulation of transcription in Saccharomyces cerevisiae and Escherichia coli.

Abstract

The focus of this work is to determine the molecular mechanisms involved in rRNA synthesis. More specifically, this research determined which component of the RNAP I transcription complex is involved in the down-regulation of rDNA transcription as yeast cells enter stationary phase. A tagged chromosomal copy of one of the subunits of the RNAP I enzyme complex will enable the RNAP I complex to be affinity purified and identified from both regulated and unregulated cultures with the hopes of elucidating the specific target of the regulatory mechanism.

It has been appreciated for a number of years that the rate of ribosomal RNA (rRNA) gene transcription is altered in response to the need for ribosome production. A key step in this process is the regulation of synthesis of ribosomal RNA. However, the regulatory mechanisms involved in transcription of rRNA remain largely uncharacterized. RNAP I specific transcription has been down-regulated in yeast which had been subjected to various environmental conditions such as starvation for an essential amino acid or nutrient, or upon entry into stationary phase. Upon reversal of these conditions the level of rRNA synthesis returned to high levels. The RNAP I activity was restored to these inactive extracts by the addition of a chromatographic fraction, from an active extract, which was enriched in the polymerase. This fraction was able to restore this RNAP I specific activity under all conditions tested. These results indicate that the polymerase or something loosely associated with the polymerase is the target of the regulatory mechanism.