Ribosomal 18S rRNA base pairs with mRNA during eukaryotic translation initiation.

Eukaryotic mRNAs often contain a Kozak sequence that helps tether the ribosome to the AUG start codon. The mRNA of histone H4 (h4) does not undergo classical ribosome scanning but has evolved a specific tethering mechanism. The cryo-EM structure of the rabbit ribosome complex with mouse h4 shows that the mRNA forms a folded, repressive structure at the mRNA entry site on the 40S subunit next to the tip of helix 16 of 18S ribosomal RNA (rRNA).

Rapid purification of ribosomal particles assembled on histone H4 mRNA: a new method based on mRNA-DNA chimaeras.

Detailed knowledge of the structure of the ribosomal particles during their assembly on mRNA is a prerequisite for understanding the intricate translation initiation process. In vitro preparation of eukaryotic translation initiation complexes is limited by the rather tricky assembly from individually purified ribosomal subunits, initiation factors and initiator tRNA. In order to directly isolate functional complexes from living cells, methods based on affinity tags have been developed which, however, often suffer from non-specific binding of proteins and/or RNAs.

Cap-assisted internal initiation of translation of histone H4.

In eukaryotes, a crucial step of translation initiation is the binding of the multifactor complex eIF4F to the 5' end of the mRNA, a prerequisite to recruitment of the activated small ribosomal 43S particle. Histone H4 mRNAs have short 5'UTRs, which do not conform to the conventional scanning-initiation model. Here we show that the ORF of histone mRNA contains two structural elements critical for translation initiation.

Deciphering membrane insertion of the diphtheria toxin T domain by specular neutron reflectometry and solid-state NMR spectroscopy.

Insertion and translocation of soluble proteins into and across biological membranes are involved in many physiological and pathological processes, but remain poorly understood. Here, we describe the pH-dependent membrane insertion of the diphtheria toxin T domain in lipid bilayers by specular neutron reflectometry and solid-state NMR spectroscopy. We gained unprecedented structural resolution using contrast-variation techniques that allow us to propose a sequential model of the membrane-insertion process at angstrom resolution along the perpendicular axis of the membrane.

Side chain resonances in static oriented proton-decoupled 15N solid-state NMR spectra of membrane proteins.

Proton-decoupled (15)N solid-state NMR spectra are used to analyze the structure, dynamics, and membrane topology of proteins uniformly labeled with (15)N. Preparation of the proteins by bacterial overexpression results in the labeling not only of the backbone amides but also of nitrogens localized within the side chains of arginine, glutamine, tryptophan, asparagines, lysines, and histidines. Most of these side chain resonances appear in the spectral region of the anisotropic backbone amides, and residual intensities have been observed also in cross-polarization spectra.

Production and isotope labeling of antimicrobial peptides in Escherichia coli by means of a novel fusion partner that enables high-yield insoluble expression and fast purification.

A method is presented that allows efficient production of antimicrobial peptides in bacteria by means of fusion to the histone fold domain of the human transcription factor TAF12. This small fusion partner drives high-level expression of peptides and leads to their accumulation in an entirely insoluble form, thereby eliminating toxicity to the host. Using the antimicrobial peptide LAH4 as an example, we demonstrate that neither affinity purification of the TAF12 fusion protein nor initial solubilization of inclusion bodies in denaturing buffers is required.

Self-promoted cellular uptake of peptide/DNA transfection complexes.

The designed alpha-helical amphipathic peptide LAH4 assembles several properties, which makes it an interesting candidate as a gene-delivery vehicle. Besides being short and soluble in aqueous solutions, LAH4 presents cationic residues, which allow for efficient complexation of DNA. In addition, this peptide is poorly hemolytic at neutral pH, while it is able to destabilize biological membranes in acidic conditions.