Activation of Transcription by Metabolic Intermediates of the Pyrimidine Biosynthetic Pathway

Abstract

Saccharomyces cerevisiae responds to pyrimidine starvation by increasing the expression of four URA genes, encoding the enzymes of de novo pyrimidine biosynthesis, three- to eightfold. The increase in gene expression is dependent on a transcriptional activator protein, Ppr1p. Here, we investigate the mechanism by which the transcriptional activity of Ppr1p responds to the level of pyrimidine biosynthetic intermediates. We find that purified Ppr1p is unable to promote activation of transcription in an in vitro system. Transcriptional activation by Ppr1p can be observed, however, if either dihydroorotic acid (DHO) or orotic acid (OA) is included in the transcription reactions. The transcriptional activation function and the DHO/OA-responsive element of Ppr1p localize to the carboxyl-terminal 134 amino acids of the protein. Thus, Ppr1p directly senses the level of early pyrimidine biosynthetic intermediates within the cell and activates the expression of genes encoding proteins required later in the pathway. These results are discussed in terms of (i) regulation of the pyrimidine biosynthetic pathway and (ii) a novel mechanism of regulating gene expression.

ACKNOWLEDGMENTS

We thank Josh Brickman, Stanley Liang, and Ronen Marmorstein for the gifts of plasmids; Judith Stanway, Ian Taylor, and Rob Hockney, Zeneca Pharmaceuticals, for help and assistance in making proteins in insect cells; and Adam Platt, Cristina Merlotti, and Ronen Marmorstein for carefully reading the manuscript.

This work was supported by grants from the Biotechnology and Biological Sciences Research Council to R.J.R.