Rapid Evolution of Sex Pheromone-Producing Enzyme Expression in Drosophila

Rapid evolution of gene expression patterns responsible for pheromone production in 24 species of Drosophila was mapped to simple mutations within the regulatory domain of the desatF gene

Kinetic CRAC uncovers a role for Nab3 in determining gene expression profiles during stress

RNA-binding proteins play a key role in shaping gene expression profiles during stress, however, little is known about the dynamic nature of these interactions and how this influences the kinetics of gene expression. To address this, we developed kinetic cross-linking and analysis of cDNAs (\u3c7CRAC), an ultraviolet cross-linking method that enabled us to quantitatively measure the dynamics of protein\u2013RNA interactions in vivo on a minute time-scale. Here, using \u3c7CRAC we measure the global RNA-binding dynamics of the yeast transcription termination factor Nab3 in response to glucose starvation. These measurements reveal rapid changes in protein\u2013RNA interactions within 1\u2009min following stress imposition. Changes in Nab3 binding are largely independent of alterations in transcription rate during the early stages of stress response, indicating orthogonal transcriptional control mechanisms. We also uncover a function for Nab3 in dampening expression of stress-responsive genes. \u3c7CRAC has the potential to greatly enhance our understanding of in vivo dynamics of protein\u2013RNA interactions

Genome-Wide Fitness and Expression Profiling Implicate Mga2 in Adaptation to Hydrogen Peroxide

Caloric restriction extends lifespan, an effect once thought to involve attenuation of reactive oxygen species (ROS) generated by aerobic metabolism. However, recent evidence suggests that caloric restriction may in fact raise ROS levels, which in turn provides protection from acute doses of oxidant through a process called adaptation. To shed light on the molecular mechanisms of adaptation, we designed a series of genome-wide deletion fitness and mRNA expression screens to identify genes involved in adaptation to hydrogen peroxide. Combined with known transcriptional interactions, the integrated data implicate Yap1 and Skn7 as central transcription factors of both the adaptive and acute oxidative responses. They also identify the transcription factors Mga2 and Rox1 as active exclusively in the adaptive response and show that Mga2 is essential for adaptation. These findings are striking because Mga2 and Rox1 have been thought to control the response to hypoxic, not oxidative, conditions. Expression profiling of mga2Δ and rox1Δ knockouts shows that these factors most strongly regulate targets in ergosterol, fatty-acid, and zinc metabolic pathways. Direct quantitation of ergosterol reveals that its basal concentration indeed depends on Mga2, but that Mga2 is not required for the decrease in ergosterol observed during adaptation

Access of the substrate to the active site of squalene and oxidosqualene cyclases: comparative inhibition, site-directed mutagenesis and homology-modelling studies

Abstract Substrate access to the active-site cavity of squalene-hopene cyclase from Alicyclobacillus acidocaldarious and lanosterol synthase [OSC (oxidosqualene cyclase)] from Saccharomyces cerevisiae was studied by an inhibition, mutagenesis and homology-modelling approach. Crystal structure and homology modelling indicate that both enzymes possess a narrow constriction that separates an entrance lipophilic channel from the active-site cavity. The role of the constriction as a mobile gate that permits substrate passage was investigated by experiments in which critically located Cys residues, either present in native protein or inserted by site-directed mutagenesis, were labelled with specifically designed thiol-reacting molecules. Some amino acid residues of the yeast enzyme, selected on the basis of sequence alignment and a homology model, were individually replaced by residues bearing side chains of different lengths, charges or hydrophobicities. In some of these mutants, substitution severely reduced enzymatic activity and thermal stability. Homology modelling revealed that in these mutants some critical stabilizing interactions could no longer occur. The possible critical role of entrance channel and constriction in specific substrate recognition by eukaryotic OSC is discussed. In cholesterol and ergosterol biosynthesis, the most significant structural alteration occurring along the pathway, that is generation of the steroid nucleus after assembly of the triterpene backbone, is brought about by lanosterol synthase. Lanosterol synthase belongs to the large family of OSCs (oxidosqualene cyclases), eukaryotic enzymes that catalyse the cyclization of 2,3-oxidosqualene into different cyclic compounds: lanosterol alone in non-photosynthetic organisms (fungi and mammals), cycloartenol, precursor of phytosterols and other tetra-and pentacyclic compounds in plants Prokaryotes possess an enzyme similar to OSCs: SHC (squalene-hopene cyclase) that converts squalene into hopene or diplopterol, pentacyclic precursors of hopanoids Squalene and OSCs catalyse among the most fascinating and complex monoenzymatic reaction

Enzymatic synthesis of an indole diterpene by an oxidosqualene cyclase: mechanistic, biosynthetic, and phylogenetic implications

Petromindole (1) is an unusual indole diterpene that bears a triterpene-like carbon skeleton, suggesting biogenesis from 3-(-oxido-geranylgeranyl)indole (4). We found that lupeol synthase (LUP1) from Arabidopsis thaliana cyclizes 4 to 1. Chiral HPLC comparisons of racemic 1 (from biomimetic cyclization of N-pivaloyl-4) with the LUP1 product and authentic 1 established the absolute stereochemistry of petromindole (3S) as that of cyclic triterpenes. Quantum mechanical calculations and conformational analysis of intermediates in the cyclization of 4 to 1 indicated that petromindole biosynthesis differs fundamentally from that of other indole diterpenes. This analysis revealed that radarins also originate from cyclization of 4 but undergo a backbone rearrangement rather than annulation to indole. The combined results support our hypothesis that native fungal petromindole synthase evolved from a pentacyclic triterpene synthase distant from most other indole diterpene synthases