Tuning transcriptional regulation through signaling: A predictive theory of allosteric induction

Allosteric regulation is found across all domains of life, yet we still lack simple, predictive theories that directly link the experimentally tunable parameters of a system to its input-output response. To that end, we present a general theory of allosteric transcriptional regulation using the Monod-Wyman-Changeux model. We rigorously test this model using the ubiquitous simple repression motif in bacteria by first predicting the behavior of strains that span a large range of repressor copy numbers and DNA binding strengths and then constructing and measuring their response. Our model not only accurately captures the induction profiles of these strains but also enables us to derive analytic expressions for key properties such as the dynamic range and $[EC_{50}]$. Finally, we derive an expression for the free energy of allosteric repressors which enables us to collapse our experimental data onto a single master curve that captures the diverse phenomenology of the induction profiles.Comment: Substantial revisions for resubmission (3 new figures, significantly elaborated discussion); added Professor Mitchell Lewis as another author for his continuing contributions to the projec

The Energetics of Molecular Adaptation in Transcriptional Regulation

Mutation is a critical mechanism by which evolution explores the functional landscape of proteins. Despite our ability to experimentally inflict mutations at will, it remains difficult to link sequence-level perturbations to systems-level responses. Here, we present a framework centered on measuring changes in the free energy of the system to link individual mutations in an allosteric transcriptional repressor to the parameters which govern its response. We find the energetic effects of the mutations can be categorized into several classes which have characteristic curves as a function of the inducer concentration. We experimentally test these diagnostic predictions using the well-characterized LacI repressor of Escherichia coli, probing several mutations in the DNA binding and inducer binding domains. We find that the change in gene expression due to a point mutation can be captured by modifying only a subset of the model parameters that describe the respective domain of the wild-type protein. These parameters appear to be insulated, with mutations in the DNA binding domain altering only the DNA affinity and those in the inducer binding domain altering only the allosteric parameters. Changing these subsets of parameters tunes the free energy of the system in a way that is concordant with theoretical expectations. Finally, we show that the induction profiles and resulting free energies associated with pairwise double mutants can be predicted with quantitative accuracy given knowledge of the single mutants, providing an avenue for identifying and quantifying epistatic interactions.Comment: 11 pages, 6 figures, supplemental info. available via http://rpgroup.caltech.edu/mwc_mutant

Development and Function of the Voltage-Gated Sodium Current in Immature Mammalian Cochlear Inner Hair Cells

Inner hair cells (IHCs), the primary sensory receptors of the mammalian cochlea, fire spontaneous Ca2+ action potentials before the onset of hearing. Although this firing activity is mainly sustained by a depolarizing L-type (CaV1.3) Ca2+ current (ICa), IHCs also transiently express a large Na+ current (INa). We aimed to investigate the specific contribution of INa to the action potentials, the nature of the channels carrying the current and whether the biophysical properties of INa differ between low- and high-frequency IHCs. We show that INa is highly temperature-dependent and activates at around −60 mV, close to the action potential threshold. Its size was larger in apical than in basal IHCs and between 5% and 20% should be available at around the resting membrane potential (−55 mV/−60 mV). However, in vivo the availability of INa could potentially increase to >60% during inhibitory postsynaptic potential activity, which transiently hyperpolarize IHCs down to as far as −70 mV. When IHCs were held at −60 mV and INa elicited using a simulated action potential as a voltage command, we found that INa contributed to the subthreshold depolarization and upstroke of an action potential. We also found that INa is likely to be carried by the TTX-sensitive channel subunits NaV1.1 and NaV1.6 in both apical and basal IHCs. The results provide insight into how the biophysical properties of INa in mammalian cochlear IHCs could contribute to the spontaneous physiological activity during cochlear maturation in vivo

An allosteric theory of transcription factor induction

Allosteric molecules serve as regulators of cellular activity across all domains of life. We present a general theory of allosteric transcriptional regulation that permits quantitative predictions for how physiological responses are tuned to environmental stimuli. To test the model's predictive power, we apply it to the specific case of the ubiquitous simple repression motif in bacteria. We measure the fold-change in gene expression at different inducer concentrations in a collection of strains that span a range of repressor copy numbers and operator binding strengths. After inferring the inducer dissociation constants using data from one of these strains, we show the broad reach of the model by predicting the induction profiles of all other strains. Finally, we derive an expression for the free energy of allosteric transcription factors which enables us to collapse the data from all of our experiments onto a single master curve, capturing the diverse phenomenology of the induction profiles

The Energetics of Molecular Adaptation in Transcriptional Regulation

Mutation is a critical mechanism by which evolution explores the functional landscape of proteins. Despite our ability to experimentally inflict mutations at will, it remains difficult to link sequence-level perturbations to systems-level responses. Here, we present a framework centered on measuring changes in the free energy of the system to link individual mutations in an allosteric transcriptional repressor to the parameters which govern its response. We find the energetic effects of the mutations can be categorized into several classes which have characteristic curves as a function of the inducer concentration. We experimentally test these diagnostic predictions using the well-characterized LacI repressor of Escherichia coli, probing several mutations in the DNA binding and inducer binding domains. We find that the change in gene expression due to a point mutation can be captured by modifying only a subset of the model parameters that describe the respective domain of the wild-type protein. These parameters appear to be insulated, with mutations in the DNA binding domain altering only the DNA affinity and those in the inducer binding domain altering only the allosteric parameters. Changing these subsets of parameters tunes the free energy of the system in a way that is concordant with theoretical expectations. Finally, we show that the induction profiles and resulting free energies associated with pairwise double mutants can be predicted with quantitative accuracy given knowledge of the single mutants, providing an avenue for identifying and quantifying epistatic interactions

Predictive shifts in free energy couple mutations to their phenotypic consequences

Mutation is a critical mechanism by which evolution explores the functional landscape of proteins. Despite our ability to experimentally inflict mutations at will, it remains difficult to link sequence-level perturbations to systems-level responses. Here, we present a framework centered on measuring changes in the free energy of the system to link individual mutations in an allosteric transcriptional repressor to the parameters which govern its response. We find that the energetic effects of the mutations can be categorized into several classes which have characteristic curves as a function of the inducer concentration. We experimentally test these diagnostic predictions using the well-characterized LacI repressor of Escherichia coli, probing several mutations in the DNA binding and inducer binding domains. We find that the change in gene expression due to a point mutation can be captured by modifying only the model parameters that describe the respective domain of the wild-type protein. These parameters appear to be insulated, with mutations in the DNA binding domain altering only the DNA affinity and those in the inducer binding domain altering only the allosteric parameters. Changing these subsets of parameters tunes the free energy of the system in a way that is concordant with theoretical expectations. Finally, we show that the induction profiles and resulting free energies associated with pairwise double mutants can be predicted with quantitative accuracy given knowledge of the single mutants, providing an avenue for identifying and quantifying epistatic interactions

Global Drivers on Southern Ocean Ecosystems: Changing Physical Environments and Anthropogenic Pressures in an Earth System

Copyright © 2020 Morley, Abele, Barnes, Cárdenas, Cotté, Gutt, Henley, Höfer, Hughes, Martin, Moffat, Raphael, Stammerjohn, Suckling, Tulloch, Waller and Constable. The manuscript assesses the current and expected future global drivers of Southern Ocean (SO) ecosystems. Atmospheric ozone depletion over the Antarctic since the 1970s, has been a key driver, resulting in springtime cooling of the stratosphere and intensification of the polar vortex, increasing the frequency of positive phases of the Southern Annular Mode (SAM). This increases warm air-flow over the East Pacific sector (Western Antarctic Peninsula) and cold air flow over the West Pacific sector. SAM as well as El Niño Southern Oscillation events also affect the Amundsen Sea Low leading to either positive or negative sea ice anomalies in the west and east Pacific sectors, respectively. The strengthening of westerly winds is also linked to shoaling of deep warmer water onto the continental shelves, particularly in the East Pacific and Atlantic sectors. Air and ocean warming has led to changes in the cryosphere, with glacial and ice sheet melting in both sectors, opening up new ice free areas to biological productivity, but increasing seafloor disturbance by icebergs. The increased melting is correlated with a salinity decrease particularly in the surface 100 m. Such processes could increase the availability of iron, which is currently limiting primary production over much of the SO. Increasing CO2 is one of the most important SO anthropogenic drivers and is likely to affect marine ecosystems in the coming decades. While levels of many pollutants are lower than elsewhere, persistent organic pollutants (POPs) and plastics have been detected in the SO, with concentrations likely enhanced by migratory species. With increased marine traffic and weakening of ocean barriers the risk of the establishment of non-indigenous species is increased. The continued recovery of the ozone hole creates uncertainty over the reversal in sea ice trends, especially in the light of the abrupt transition from record high to record low Antarctic sea ice extent since spring 2016. The current rate of change in physical and anthropogenic drivers is certain to impact the Marine Ecosystem Assessment of the Southern Ocean (MEASO) region in the near future and will have a wide range of impacts across the marine ecosystem