In vivo tmRNA protection by SmpB and pre-ribosome binding conformation in solution.

TmRNA is an abundant RNA in bacteria with tRNA and mRNA features. It is specialized in trans-translation, a translation rescuing system. We demonstrate that its partner protein SmpB binds the tRNA-like region (TLD) in vivo and chaperones the fold of the TLD-H2 region. We use an original approach combining the observation of tmRNA degradation pathways in a heterologous system, the analysis of the tmRNA digests by MS and NMR, and co-overproduction assays of tmRNA and SmpB. We study the conformation in solution of tmRNA alone or in complex with one SmpB before ribosome binding using SAXS. Our data show that Mg(2+) drives compaction of the RNA structure and that, in the absence of Mg(2+), SmpB has a similar effect albeit to a lesser extent. Our results show that tmRNA is intrinsically structured in solution with identical topology to that observed on complexes on ribosomes which should facilitate its subsequent recruitment by the 70S ribosome, free or preloaded with one SmpB molecule.

Characterization of monomeric intermediates during VSV glycoprotein structural transition.

Entry of enveloped viruses requires fusion of viral and cellular membranes, driven by conformational changes of viral glycoproteins. Crystal structures provide static pictures of pre- and post-fusion conformations of these proteins but the transition pathway remains elusive. Here, using several biophysical techniques, including analytical ultracentrifugation, circular dichroïsm, electron microscopy and small angle X-ray scattering, we have characterized the low-pH-induced fusogenic structural transition of a soluble form of vesicular stomatitis virus (VSV) glycoprotein G ectodomain (G(th), aa residues 1-422, the fragment that was previously crystallized). While the post-fusion trimer is the major species detected at low pH, the pre-fusion trimer is not detected in solution. Rather, at high pH, G(th) is a flexible monomer that explores a large conformational space. The monomeric population exhibits a marked pH-dependence and adopts more elongated conformations when pH decreases. Furthermore, large relative movements of domains are detected in absence of significant secondary structure modification. Solution studies are complemented by electron micrographs of negatively stained viral particles in which monomeric ectodomains of G are observed at the viral surface at both pH 7.5 and pH 6.7. We propose that the monomers are intermediates during the conformational change and thus that VSV G trimers dissociate at the viral surface during the structural transition.