Unigenic evolution: A novel genetic method applied to the transcriptional regulator Gcr1p in Saccharomyces cerevisiae

Stephen Jackson Deminoff

Abstract

The product of the GCR1 gene in Saccharomyces cerevisiae complexes with the essential multifunctional regulatory protein Rap1p to activate transcription of glycolytic and translational component genes, which together make up a large fraction of the total protein in the cell. To identify functionally important domains in Gcr1p, we developed a novel genetic method that combines multiple rounds of random mutagenesis in vitro with a screen for functional alleles in vivo to generate a statistical map of regions that are underrepresented for missense mutations. Examination of the distribution of 315 point mutations in 24 variant alleles allowed the localization of four hypomutable regions in GCR1 (A, B, C, and D). Dispensable N-terminal (intronic) and C-terminal portions of the evolved region were included as controls and were, as expected, not hypomutable. The analysis of several insertion, deletion, and point mutations, combined with a comparison of the hypomutability and hydrophobicity plots of Gcr1p, suggested that some of the hypomutable regions may individually or in combination correspond to functionally important surface domains. In particular, I determined that region D contains a putative leucine zipper and is necessary and sufficient for Gcr1p homodimerization. Subsequently, we identified a match to leucine zipper motifs in Gcr2p, which forms a complex with Gcr1p that I have coimmunoprecipitated from yeast extracts. The potential leucine zipper in Gcr2p is necessary for Gcr2p function and interaction with Gcr1p. Also, a point mutation in hypomutable region A of Gcr1p leads to destabilization of Gcr1p in the absence of Gcr2p. Models for Rap 1p/Gcr1p/Gcr2p transcription will be discussed.