E-GEOD-52135 - Towards in vivo regulatory kinetics: PurR activation by PRPP during purine depletion in Lactococcus
Released on 1 December 2013, last updated on 3 June 2014
Lactococcus lactis subsp. cremoris MG1363
Short-term adaptation to changing environments relies on regulatory elements translating changing metabolite concentrations into a specifically optimized transcriptome. So far the focus of analyses has been divided between regulatory elements identified in vivo and kinetic studies of small molecules interacting with the regulatory elements in vitro. Here we describe how in vivo Regulatory Kinetics can describe a regulon through the effects of the metabolite controlling it, exemplified by temporal purine exhaustion in Lactococcus lactis. We deduced a causal relation between the pathway precursor 5-phosphoribosyl-1-pyrophosphate (PRPP) and each individual mRNA levels, whereby unambiguous and homogenous relations could be obtained for PurR regulated genes, thus linking a specific regulon to a specific metabolite. As PurR activates gene expression upon binding of PRPP, the pur mRNA curves reflect the in vivo kinetics of PurR PRPP binding and activation. The method singled out the xpt-pbuX operon as kinetically distinct, which was found to be caused by a guanine riboswitch whose regulation was overlaying the PurR regulation. The strategy outlined here can be adapted to analyze the individual effects of members from larger metabolomes in virtually any organism, for elucidating regulatory networks in vivo. Agilent 8x15k custom microarrays Fifteen samples from two cultures were taken in a time-course experiment.
transcription profiling by array
Christian Bille Jendresen <firstname.lastname@example.org>, Christian B Jendresen, Jan Martinussen, Laurent Gautier, Meng Lui, Mogens Kilstrup, Peter Dimitrov