(283b) Cellular Biosynthesis Responds to Changing Nutrient Environments: Predicting in Vivo Behavior from in Vitro Models

Living cells respond rapidly to nutrient-rich environments by enhancing their production of ribosomal RNA (rRNA), a limiting component in the biosynthesis of ribosomes. Studies of rRNA transcription by the RNA polymerase (RNAP) of Escherichia coli have identified key regulatory roles for small molecules guanosine tetraphosphate (ppGpp) and initiating nucleotide (iNTP), and, more recently, the protein DksA, a transcription factor. Most studies to date have been performed in vitro at concentrations that are useful for determining potential molecular mechanisms but are non-physiological. It is not known to what extent such findings can be used to predict cellular responses to changing nutrient environments. To address this question we have developed a mathematical model for rRNA transcriptional responses to changes in nutritional/environmental conditions. Our model accounts for binding of RNAP to its rRNA promoter to form a closed complex, isomerization from a closed complex to an open complex, reversible incorporation of the iNTP, transcript elongation, and clearance of the promoter. Further, the model incorporates interactions between ppGpp and DksA with transcription intermediates, and it includes an empirical correction to account for salt effects. The biophysical parameters of the model were determined by non-linear parameter estimation using 33 single- and multi-round transcription experiments spanning 487 in vitro measurements. By incorporating in vivo measurements of ppGpp and ATP, the model was used to predict rates of rRNA production for cellular responses to amino acid infusion (upshift), addition of an inhibitor of glucose metabolism (downshift), and dilution of stationary phase cells into fresh medium (outgrowth). Inclusion of DksA was essential for allowing the model to capture the magnitude and timing of transcriptional responses in all three cases. In summary, this work provides a foundation for using in vitro results to predict the kinetics of in vivo transcriptional responses.