Transient disome complex formation in native polysomes during ongoing protein synthesis captured by cryo-EM.

Structural studies of translating ribosomes traditionally rely on in vitro assembly and stalling of ribosomes in defined states. To comprehensively visualize bacterial translation, we reactivated ex vivo-derived E. coli polysomes in the PURE in vitro translation system and analyzed the actively elongating polysomes by cryo-EM. We find that 31% of 70S ribosomes assemble into disome complexes that represent eight distinct functional states including decoding and termination intermediates, and a pre-nucleophilic attack state. The functional diversity of disome complexes together with RNase digest experiments suggests that paused disome complexes transiently form during ongoing elongation. Structural analysis revealed five disome interfaces between leading and queueing ribosomes that undergo rearrangements as the leading ribosome traverses through the elongation cycle. Our findings reveal at the molecular level how bL9's CTD obstructs the factor binding site of queueing ribosomes to thwart harmful collisions and illustrate how translation dynamics reshape inter-ribosomal contacts.

Structure of the ribosome-bound cricket paralysis virus IRES RNA.

Internal ribosome entry sites (IRESs) facilitate an alternative, end-independent pathway of translation initiation. A particular family of dicistroviral IRESs can assemble elongation-competent 80S ribosomal complexes in the absence of canonical initiation factors and initiator transfer RNA. We present here a cryo-EM reconstruction of a dicistroviral IRES bound to the 80S ribosome. The resolution of the cryo-EM reconstruction, in the subnanometer range, allowed the molecular structure of the complete IRES in its active, ribosome-bound state to be solved. The structure, harboring three pseudoknot-containing domains, each with a specific functional role, shows how defined elements of the IRES emerge from a compactly folded core and interact with the key ribosomal components that form the A, P and E sites, where tRNAs normally bind. Our results exemplify the molecular strategy for recruitment of an IRES and reveal the dynamic features necessary for internal initiation.

Cell wall invertase expression at the apical meristem alters floral, architectural, and reproductive traits in Arabidopsis thaliana.

Resource allocation is a major determinant of plant fitness and is influenced by external as well as internal stimuli. We have investigated the effect of cell wall invertase activity on the transition from vegetative to reproductive growth, inflorescence architecture, and reproductive output, i.e. seed production, in the model plant Arabidopsis thaliana by expressing a cell wall invertase under a meristem-specific promoter. Increased cell wall invertase activity causes accelerated flowering and an increase in seed yield by nearly 30%. This increase is caused by an elevation of the number of siliques, which results from enhanced branching of the inflorescence. On the contrary, as cytosolic enzyme, the invertase causes delayed flowering, reduced seed yield, and branching. This demonstrates that invertases not only are important in determining sink strength of storage organs but also play a role in regulating developmental processes.