Insights into the Regulatory Networks and RNA Processes Governed by the Escherichia Coli Ribosome Associated Protein ProQ, and the Saccharomyces Cerevisiae Kinase Ksp1p.

Abstract

Cells must be able to sense external signals and respond by modulating gene expression to meet the environmental conditions. The mechanisms for combating environmental stresses are myriad. Two examples from bacteria and yeast are investigated here. During osmotic stress, E. coli respond by importing osmoprotectant molecules, such as proline, from their surroundings. The expression of the proline porter ProP is regulated both transcriptionally and translationally. ProQ regulates ProP expression at the level of translation via an undetermined mechanism. Examination of ProQ showed that it can be found associated with translating ribosomes. This interaction is likely due to a tethering of ProQ to the ribosome by the mRNA being translated; though, ProQ can bind with lowered affinity to mRNA-free ribosomal particles. ProQ was previously shown to bind RNA in vitro, but this binding is not selective for proP. In addition to affecting ProP expression, ProQ also modulates biofilm formation, suggesting a larger role for ProQ than its control of ProP translation and proline uptake. In yeast, environmental stresses sensed at the cell surface are transduced to regulate gene expression by kinases. Kinase signaling is accomplished by addition of a phosphate group to a serine, threonine, or tyrosine of a downstream target. Deletion of the yeast Ser/Thr kinase Ksp1p, as well as disruption of its kinase domain, causes a defect in the cells response to nitrogen stress, manifesting as a failure to undergo a switch from vegetative growth to pseudohyphal growth. Phosphproteomic analysis of global changes in the phosphorylation state of proteins, between a wild type and kinase-dead Ksp1p during nitrogen stress, yielded potential targets of Ksp1p. The dataset includes previously characterized phosphorylation sites, as well as novel sites and known sites of unknown function. Further analysis reveals Ksp1p signaling likely occurs via the conserved Protein Kinase A (PKA) pathway. Beyond pseudohyphal growth, potential targets of Ksp1p included known components of ribonuclear protein (RNP) aggregates called stress granules. Point mutations of implicated residues show abnormal phenotypes for pseudohyphal growth and/or RNP formation. Together the data position Ksp1p in the PKA pathway to govern changes in cell morphology and RNP formation.