The mechanisms by which eukaryotes regulate gene expression are important for understanding many complex biological phenomena including human diseases. Prevention and treatment of such diseases have been and will continue to be improved by basic knowledge of gene regulation, especially because molecular mechanisms of transcriptional initiation are highly conserved in eukaryotic organisms ranging from human to yeast. This proposal will continue to investigate basic issues concerning molecular mechanisms of transcriptional regulation, polyadenylation, and mRNA stability in yeast, by combining molecular genetic, biochemical, functional genomic, and evolutionary approaches. Work in the first two sections will take advantage of our novel and recently developed methodologies for measuring half-lives, structure (DREADS via chemical probes), protein binding (CLIP-READS), and poly(A) length (A-READS) of individual mRNA 3' isoforms. First, in the area of polyadenylation, we will A) address the mechanisms for why polyadenylation is restricted to the 3' UTR. B) identify factors that are responsible for the wild-type poly(A) pattern, C) determine the factors and mechanistic basis for regulated polyadenylation during the diauxic shift (and perhaps other conditions), and D) elucidate 3'-isoform variation and regulation of poly(A) length. Second, for studies of mRNA stabilization/destabilization elements and half-lives of 3' isoforms and, we will A) perform RNA structural analysis during the degradation process, B) identify protein factors mediating the large differences in mRNA isoform stabilities C) perform directed genetic experiments to address how secondary structure affects mRNA stability, D) identify mRNA stabilization and destabilizing elements that differentially affected by environmental conditions, and E) identify factors important for regulated mRNA half-lives, which the goal of elucidating the mechanism of regulated mRNA stability. Third, we will address a variety of issues concerning transcriptional regulation including A) the nature of the transcriptional activator that coordinately regulates ribosomal protein gene expression via recruitment of TFIID, B) determining the mechanistic basis of why activator proteins do not function when bound downstream of or far away from the core promoter, C) DNA looping mechanisms, particularly the nature of the protein-protein interactions needed to form the loop and to stimulate transcription, and D) examining the role of histone acetylation in transcriptional regulation by generating non-acetylable derivatives of the 4 histones. Fourth, we will use a novel conceptual and experimental approach to distinguish biological function from biological noise that is based on a comparison of physiological responses, RNA and transcription factor binding profiles, and effects of mutations in yeast species of varying evolutionary distance. We will explicitly measure biological noise by making functional measurements of evolutionary irrelevant or random-sequence DNA in yeast. Overall, the proposal will answer fundamental questions about the interlinked processes of transcription, polyadenylation, and mRNA stability in a mechanistic and evolutionary framework.
Regulation of gene expression is a critical aspect of many biological phenomena (e.g. cell growth, development of multicellular organisms, the response to environmental conditions, and evolution), and alteration of normal gene regulation can lead to human disease. This proposal investigates fundamental molecular mechanisms of gene regulation at several levels, and it also uses a novel functional evolutionary approach to understand broad issues related to biological function. The results will continue to shed new light on fundamental issues in gene regulation and will have a significant impact on our understanding of human biology and disease.
