Structural Analysis of the CDK-Cyclin Complex of Pho85-Pho80 and Genome-Wide Characterization of the Phosphate Starvation Response in Schizosaccharomyces pombe
Carter-O'Connell, Ian O’Brien
MetadataShow full item record
CitationCarter-O'Connell, Ian O’Brien. 2012. Structural Analysis of the CDK-Cyclin Complex of Pho85-Pho80 and Genome-Wide Characterization of the Phosphate Starvation Response in Schizosaccharomyces pombe. Doctoral dissertation, Harvard University.
AbstractInorganic phosphate is an essential nutrient required by all organisms for optimal growth. During phosphate starvation, Saccharomyces cerevisiae induces a set of genes responsible for the regulation of inorganic phosphate acquisition. The phosphate-responsive signaling (PHO) pathway controls this response, with the CDK-cyclin complex Pho85-Pho80 playing a prominent role. Here we report the X-ray structure of the Pho85-Pho80 complex, identifying the unique structural features that distinguish it from other cell cycle associated CDK-cyclin complexes. The structure reveals a specific salt bridge between a Pho85 arginine and a Pho80 aspartate that maintains a Pho80 loop confirmation important for substrate recognition and makes phosphorylation of the Pho85 activation loop dispensible. We show that a cluster of residues distal to the kinase active site are involved in a high affinity interaction between the Pho80 cyclin and the transcription factor substrate (Pho4). The structure also reveals a separate high affinity binding site for the CDK inhibitor (Pho81). The fission yeast, Schizosaccharomyces pombe, regulates expression of the secreted acid phosphatase \((pho1^+)\) via a non-orthologous PHO pathway. The genes induced by phosphate limitation and the molecular mechanism by which the genetically identified positive \((pho7^+)\) and negative \((csk1^+)\) regulators function are not known. Here we use a combination of molecular biology, expression microarrays, chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq), and global transcriptome sequencing (RNA-Seq) to characterize the role of \(pho7^+\) and \(csk1^+\) in the PHO response. We show that there is a fast and slow response to phosphate starvation, each with defined regulatory roles. We use ChIP-Seq to identify members of the Pho7 regulon and characterize Pho7 binding dynamics in response to phosphate-limitation and Csk1 activity. We identify a conserved PHO response for the PHO5 \((pho1^+)\), PHO84 \((spbc8e4.01c^+)\), and GIT1 \((spbc1271.09^+)\) orthologs. We show that activation of \(pho1^+\) requires Pho7 binding to a UAS in the \(pho1^+\) promoter and that a URS is necessary for Csk1 repression. We find that Pho7-dependent activation is not limited to phosphate-starvation, as additional environmental stress response pathways require \(pho7^+\) for maximal induction. Using RNA-Seq we show that Pho7 is also involved in regulating non-coding transcription and is a bi-functional transcription factor.
Citable link to this pagehttp://nrs.harvard.edu/urn-3:HUL.InstRepos:9414562
- FAS Theses and Dissertations