Jacques P. Bothma

Dynamic regulation of eve stripe 2 expression reveals transcriptional bursts in living Drosophila embryos.

Significance There is considerable information about the spatial regulation of gene expression during pattern formation in animal development. Significantly less is known about temporal control, in part due to our inability to analyze gene activity in real time. Using a recently developed approach for the visualization of gene expression in living Drosophila embryos, we examined the well-known even-skipped stripe 2 expression pattern. Surprisingly, we observe that this classic pattern is quite transient and generated by discontinuous surges of transcriptional activity in individual cells. These results challenge a purely static framework for dissecting developmental programs and emphasize the importance of the dynamic features of pattern formation. We present the use of recently developed live imaging methods to examine the dynamic regulation of even-skipped (eve) stripe 2 expression in the precellular Drosophila embryo. Nascent transcripts were visualized via MS2 RNA stem loops. The eve stripe 2 transgene exhibits a highly dynamic pattern of de novo transcription, beginning with a broad domain of expression during nuclear cycle 12 (nc12), and progressive refinement during nc13 and nc14. The mature stripe 2 pattern is surprisingly transient, constituting just ∼15 min of the ∼90-min period of expression. Nonetheless, this dynamic transcription profile faithfully predicts the limits of the mature stripe visualized by conventional in situ detection methods. Analysis of individual transcription foci reveals intermittent bursts of de novo transcription, with duration cycles of 4–10 min. We discuss a multistate model of transcription regulation and speculate on its role in the dynamic repression of the eve stripe 2 expression pattern during development.

Long-Range Intra-Protein Communication Can Be Transmitted by Correlated Side-Chain Fluctuations Alone

Allosteric regulation is a key component of cellular communication, but the way in which information is passed from one site to another within a folded protein is not often clear. While backbone motions have long been considered essential for long-range information conveyance, side-chain motions have rarely been considered. In this work, we demonstrate their potential utility using Monte Carlo sampling of side-chain torsional angles on a fixed backbone to quantify correlations amongst side-chain inter-rotameric motions. Results indicate that long-range correlations of side-chain fluctuations can arise independently from several different types of interactions: steric repulsions, implicit solvent interactions, or hydrogen bonding and salt-bridge interactions. These robust correlations persist across the entire protein (up to 60 Å in the case of calmodulin) and can propagate long-range changes in side-chain variability in response to single residue perturbations.

Enhancer additivity and non-additivity are determined by enhancer strength in the Drosophila embryo.

Metazoan genes are embedded in a rich milieu of regulatory information that often includes multiple enhancers possessing overlapping activities. In this study, we employ quantitative live imaging methods to assess the function of pairs of primary and shadow enhancers in the regulation of key patterning genes-knirps, hunchback, and snail-in developing Drosophila embryos. The knirps enhancers exhibit additive, sometimes even super-additive activities, consistent with classical gene fusion studies. In contrast, the hunchback enhancers function sub-additively in anterior regions containing saturating levels of the Bicoid activator, but function additively in regions where there are diminishing levels of the Bicoid gradient. Strikingly sub-additive behavior is also observed for snail, whereby removal of the proximal enhancer causes a significant increase in gene expression. Quantitative modeling of enhancer–promoter interactions suggests that weakly active enhancers function additively while strong enhancers behave sub-additively due to competition with the target promoter. DOI: http://dx.doi.org/10.7554/eLife.07956.001

The role of quantum effects in proton transfer reactions in enzymes: Quantum tunneling in a noisy environment?

We consider the role of quantum effects in the transfer of hydrogen-like species in enzyme-catalyzed reactions. This review is stimulated by claims that the observed magnitude and temperature dependence of kinetic isotope effects (KIEs) implies that quantum tunneling below the energy barrier associated with the transition state significantly enhances the reaction rate in many enzymes. We review the path integral approach and the Caldeira–Leggett model, which provides a general framework to describe and understand tunneling in a quantum system that interacts with a noisy environment at nonzero temperature. Here the quantum system is the active site of the enzyme, and the environment is the surrounding protein and water. Tunneling well below the barrier only occurs for temperatures less than a temperature T0, which is determined by the curvature of the potential energy surface near the top of the barrier. We argue that for most enzymes this temperature is less than room temperature. We review typical values for the parameters in the Caldeira–Leggett Hamiltonian, including the frequency-dependent friction and noise due to the environment. For physically reasonable parameters, we show that quantum transition state theory gives a quantitative description of the temperature dependence and magnitude of KIEs for two classes of enzymes that have been claimed to exhibit signatures of quantum tunneling. The only quantum effects are those associated with the transition state, both reflection at the barrier top and tunneling just below the barrier. We establish that the friction and noise due to the environment are weak and only slightly modify the reaction rate. Furthermore, at room temperature and for typical energy barriers environmental fluctuations with frequencies much less than 1000 cm−1 do not have a significant effect on quantum corrections to the reaction rate. This is essentially because the time scales associated with the dynamics of proton transfer are faster than much of the low-frequency noise associated with the protein and solvent.We consider the role of quantum effects in the transfer of hyrogen-like species in enzyme-catalysed reactions. This study is stimulated by claims that the observed magnitude and temperature dependence of kinetic isotope effects imply that quantum tunneling below the energy barrier associated with the transition state significantly enhances the reaction rate in many enzymes. We use a path integral approach which provides a general framework to understand tunneling in a quantum system which interacts with an environment at non-zero temperature. Here the quantum system is the active site of the enzyme and the environment is the surrounding protein and water. Tunneling well below the barrier only occurs for temperatures less than a temperature T0 which is determined by the curvature of potential energy surface near the top of the barrier. We argue that for most enzymes this temperature is less than room temperature. For physically reasonable parameters quantum transition state theory gives a quantitative description of the temperature dependence and magnitude of kinetic isotope effects for two classes of enzymes which have been claimed to exhibit signatures of quantum tunneling. The only quantum effects are those associated with the transition state, both reflection at the barrier top and tunneling just below the barrier. We establish that the friction due to the environment is weak and only slightly modifies the reaction rate. Furthermore, at room temperature and for typical energy barriers environmental degrees of freedom with frequencies much less than 1000 cm do not have a significant effect on quantum corrections to the reaction rate.

Morphogen Gradients: Limits to Signaling or Limits to Measurement?

In Drosophila embryos, a concentration gradient of nuclear Dorsal protein controls pattern formation along the dorsal-ventral axis. Recent quantitative studies agree on the temporal dynamics of the gradient, but disagree on its spatial limits.

Development: Lights, Camera, Action — The Drosophila Embryo Goes Live!

Live imaging of developmental gene expression in Drosophila embryos opens up exciting new prospects for understanding gene regulation during development.