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Abstract Fast mixing of small volumes of solutions in microfluidic devices is essential for an accurate control and observation of the dynamics of a reaction in biological or chemical studies. It is often, however, a challenging task, as the Reynolds number (Re) in microscopic devices is typically

Sample preparation on cryo-EM grids can give various results, from very thin ice and homogeneous particle distribution (ideal case) to unwanted behavior such as particles around the “holes” or complexes that do not entirely correspond to the one in solution (real life). We recently run into such a case and finally found out that variations in the 3D reconstructions were systematically correlated with the grid batches that were used. We report the use of several techniques to investigate the grids' characteristics, namely TEM, SEM, Auger spectroscopy and Infrared Interferometry. This allowed us to diagnose the origin of grid preparation problems and to adjust glow discharge parameters. The methods used for each approach are described and the results obtained on a common specific case are reported.

Translation initiation (TI) allows accurate selection of the initiation codon on a messenger RNA (mRNA) and defines the reading frame. In all domains of life, translation initiation generally occurs within a macromolecular complex made up of the small ribosomal subunit, the mRNA, a specialized methionylated initiator tRNA, and translation initiation factors (IFs). Once the start codon is selected at the P site of the ribosome and the large subunit is associated, the IFs are released and a ribosome competent for elongation is formed. However, even if the general principles are the same in the three domains of life, the molecular mechanisms are different in bacteria, eukaryotes, and archaea and may also vary depending on the mRNA. Because TI mechanisms have evolved lately, their studies bring important information about the evolutionary relationships between extant organisms. In this context, recent structural data on ribosomal complexes and genome-wide studies are particularly valuable. This review focuses on archaeal translation initiation highlighting its relationships with either the eukaryotic or the bacterial world. Eukaryotic features of the archaeal small ribosomal subunit are presented. Ribosome evolution and TI mechanisms diversity in archaeal branches are discussed. Next, the use of leaderless mRNAs and that of leadered mRNAs having Shine-Dalgarno sequences is analyzed. Finally, the current knowledge on TI mechanisms of SD-leadered and leaderless mRNAs is detailed.

Designed enzymes are of fundamental and technological interest. Experimental directed evolution still has significant limitations, and computational approaches are a complementary route. A designed enzyme should satisfy multiple criteria: stability, substrate binding, transition state binding. Such multi-objective design is computationally challenging. Two recent studies used adaptive importance sampling Monte Carlo to redesign proteins for ligand binding. By first flattening the energy landscape of the apo protein, they obtained positive design for the bound state and negative design for the unbound. We have now extended the method to design an enzyme for specific transition state binding, i.e., for its catalytic power. We considered methionyl-tRNA synthetase (MetRS), which attaches methionine (Met) to its cognate tRNA, establishing codon identity. Previously, MetRS and other synthetases have been redesigned by experimental directed evolution to accept noncanonical amino acids as substrates, leading to genetic code expansion. Here, we have redesigned MetRS computationally to bind several ligands: the Met analog azidonorleucine, methionyl-adenylate (MetAMP), and the activated ligands that form the transition state for MetAMP production. Enzyme mutants known to have azidonorleucine activity were recovered by the design calculations, and 17 mutants predicted to bind MetAMP were characterized experimentally and all found to be active. Mutants predicted to have low activation free energies for MetAMP production were found to be active and the predicted reaction rates agreed well with the experimental values. We suggest the present method should become the paradigm for computational enzyme design.

Initiation factor eIF2, common to eukaryotes and archaea, is a central actor in translation initiation. Here the authors describe two cryo-EM structures of archaeal 30S initiation complexes that provide a novel view of the central role that e/aIF2 plays in start codon selection.

Summary: cGMP is critical to a variety of cellular processes, but the available tools to interfere with endogenous cGMP lack cellular and subcellular specificity. We introduce SponGee, a genetically encoded chelator of this cyclic nucleotide that enables in vitro and in vivo manipulations in single cells and in biochemically defined subcellular compartments. SponGee buffers physiological changes in cGMP concentration in various model systems while not affecting cAMP signals. We provide proof-of-concept strategies by using this tool to highlight the role of cGMP signaling in vivo and in discrete subcellular domains. SponGee enables the investigation of local cGMP signals in vivo and paves the way for therapeutic strategies that prevent downstream signaling activation. : Ros et al. developed SponGee, a genetically encoded cGMP chelator that enables the manipulation of this second messenger in single cells with subcellular specificity. SponGee alters the migration of developing cortical neurons in vivo. Lipid raft targeting of SponGee prevents axon repulsion, in contrast to exclusion from this subcellular compartment. Keywords: cGMP buffer, subcellular compartment, single cell pharmacology, axon guidance, neuronal migration, lipid grafts, genetically encoded, FRET, ThPDE5VV, PKG

Understanding molecular mechanisms of ribosomal translation sheds light on the emergence and evolution of protein synthesis in the three domains of life. Universally, ribosomal translation is described in three steps: initiation, elongation and termination. During initiation, a macromolecular complex assembled around the small ribosomal subunit selects the start codon on the mRNA and defines the open reading frame. In this review, we focus on the comparison of start codon selection mechanisms in eukaryotes and archaea. Eukaryotic translation initiation is a very complicated process, involving many initiation factors. The most widespread mechanism for the discovery of the start codon is the scanning of the mRNA by a pre-initiation complex until the first AUG codon in a correct context is found. In archaea, long-range scanning does not occur because of the presence of Shine-Dalgarno (SD) sequences or of short 5′ untranslated regions. However, archaeal and eukaryotic translation initiations have three initiation factors in common: e/aIF1, e/aIF1A and e/aIF2 are directly involved in the selection of the start codon. Therefore, the idea that these archaeal and eukaryotic factors fulfill similar functions within a common structural ribosomal core complex has emerged. A divergence between eukaryotic and archaeal factors allowed for the adaptation to the long-range scanning process versus the SD mediated prepositioning of the ribosome.