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Rapid and robust signaling in the CsrA cascade via RNA-protein interactions and feedback regulation.

ABSTRACT: Bacterial survival requires the rapid propagation of signals through gene networks during stress, but how this is achieved is not well understood. This study systematically characterizes the signaling dynamics of a cascade of RNA-protein interactions in the CsrA system, which regulates stress responses and biofilm formation in Escherichia coli. Noncoding RNAs are at the center of the CsrA system; target mRNAs are bound by CsrA proteins that inhibit their translation, CsrA proteins are sequestered by CsrB noncoding RNAs, and the degradation of CsrB RNAs is increased by CsrD proteins. Here, we show using in vivo experiments and quantitative modeling that the CsrA system integrates three strategies to achieve rapid and robust signaling. These strategies include: (i) the sequestration of stable proteins by noncoding RNAs, which rapidly inactivates protein activity; (ii) the degradation of stable noncoding RNAs, which enables their rapid removal; and (iii) a negative-feedback loop created by CsrA repression of CsrD production, which reduces the time for the system to achieve steady state. We also demonstrate that sequestration in the CsrA system results in signaling that is robust to growth rates because it does not rely on the slow dilution of molecules via cell division; therefore, signaling can occur even during growth arrest induced by starvation or antibiotic treatment.

SUBMITTER: Adamson DN

PROVIDER: S-EPMC3740896 | biostudies-other | 2013 Aug

REPOSITORIES: biostudies-other

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Rapid and robust signaling in the CsrA cascade via RNA-protein interactions and feedback regulation.

Adamson David Nellinger DN Lim Han N HN

Proceedings of the National Academy of Sciences of the United States of America 20130722 32

Bacterial survival requires the rapid propagation of signals through gene networks during stress, but how this is achieved is not well understood. This study systematically characterizes the signaling dynamics of a cascade of RNA-protein interactions in the CsrA system, which regulates stress responses and biofilm formation in Escherichia coli. Noncoding RNAs are at the center of the CsrA system; target mRNAs are bound by CsrA proteins that inhibit their translation, CsrA proteins are sequestere ...[more]

PMID: 23878244

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Project description:Signal output from receptor-G-protein-effector modules is a dynamic function of the nucleotide exchange activity of the receptor, the GTPase-accelerating activity of GTPase-activating proteins (GAPs), and their interactions. GAPs may inhibit steady-state signaling but may also accelerate deactivation upon removal of stimulus without significantly inhibiting output when the receptor is active. Further, some effectors (e.g., phospholipase C-beta) are themselves GAPs, and it is unclear how such effectors can be stimulated by G proteins at the same time as they accelerate G protein deactivation. The multiple combinations of protein-protein associations and interacting regulatory effects that allow such complex behaviors in this system do not permit the usual simplifying assumptions of traditional enzyme kinetics and are uniquely subject to systems-level analysis. We developed a kinetic model for G protein signaling that permits analysis of both interactive and independent G protein binding and regulation by receptor and GAP. We evaluated parameters of the model (all forward and reverse rate constants) by global least-squares fitting to a diverse set of steady-state GTPase measurements in an m1 muscarinic receptor-G(q)-phospholipase C-beta1 module in which GTPase activities were varied by approximately 10(4)-fold. We provide multiple tests to validate the fitted parameter set, which is consistent with results from the few previous pre-steady-state kinetic measurements. Results indicate that (1) GAP potentiates the GDP/GTP exchange activity of the receptor, an activity never before reported; (2) exchange activity of the receptor is biased toward replacement of GDP by GTP; (3) receptor and GAP bind G protein with negative cooperativity when G protein is bound to either GTP or GDP, promoting rapid GAP binding and dissociation; (4) GAP indirectly stabilizes the continuous binding of receptor to G protein during steady-state GTPase hydrolysis, thus further enhancing receptor activity; and (5) receptor accelerates GDP/GTP exchange primarily by opening an otherwise closed nucleotide binding site on the G protein but has minimal effect on affinity (K(assoc) = k(assoc)/k(dissoc)) of G protein for nucleotide. Model-based simulation explains how GAP activity can accelerate deactivation >10-fold upon removal of agonist but still allow high signal output while the receptor is active. Analysis of GTPase flux through distinct reaction pathways and consequent accumulation of specific GTPase cycle intermediates indicate that, in the presence of a GAP, the receptor remains bound to G protein throughout the GTPase cycle and that GAP binds primarily during the GTP-bound phase. The analysis explains these behaviors and relates them to the specific regulatory phenomena described above. The work also demonstrates the applicability of appropriately data-constrained system-level analysis to signaling networks of this scale.

Dataset Information

Rapid and robust signaling in the CsrA cascade via RNA-protein interactions and feedback regulation.

Publications

Rapid and robust signaling in the CsrA cascade via RNA-protein interactions and feedback regulation.

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