Regulation and variation of subunits of RNA polymerase II in Saccharomyces cerevisiae

grantor: University of TorontoRNA polymerase (RNAP) uses ribonucleoside triphosphates as a substrate to form RNA chains. The RNA is a faithful copy of one strand of a double-stranded DNA template along which RNAP moves while making the RNA. This process (called transcription) is highly regulated such that DNA-encoded genes are transcribed at a large variety of rates and at different times in response to cues within and outside the cell. The goal of my thesis work was to investigate the mechanisms that regulate the synthesis of RNAP in yeast cells. I focused on the form of RNAP (RNAPII) of the yeast, 'Saccharomyces cerevisiae', that is responsible primarily for transcribing genes that encode proteins. I have shown that a 10-fold reduction in the level of the largest subunit of RNAPII, and likely the level of RNAPII itself, causes slow growth, temperature-sensitivity, and the inability to grow on medium lacking inositol. Hence, the level of RNAPII must be carefully maintained for normal cell growth. I next examined elements that control the synthesis of RNAPII. I have demonstrated that the DNA sequences which are upstream of the genes encoding the two largest subunits of RNAPII, namely 'RPO21' and 'RPO22', contain binding sites for two abundant transcription factors called Abf1p and Reb1p, and thymidine-rich sequences downstream of these binding sites. Both the binding sites and the T-rich regions are important for the expression of these genes. An examination of the upstream sequences of other RNAPII subunit genes revealed binding sites for Abf1p and Reb1p as well as nearby thymidine-rich sequences. This may indicate that there is a mechanism for the coordinate synthesis of RNAPII subunit genes. I sought evidence for a feedback regulatory mechanism that may control the synthesis of RNAPII. Either the underproduction of Rpo21p, or the depletion of Fcp1p, an RNAPII phosphatase that has a critical role in transcription, leads to a 5-fold increase in the expression of a reporter gene that is controlled by 'RPO21' regulatory sequences. The increase is not observed with other subunits. I discuss the implications of these results and future directions.Ph.D