Breaking Down the Final Steps in 40S Ribosomal Subunit Assembly.

[摘要] Eukaryotic ribosome assembly requires almost 200 conserved assembly factors (AFs) that are not part of the mature ribosome. To understand the function of AFs involved in late cytoplasmic steps of 40S maturation, biochemical and structural investigations of the latest steps in 40S maturation were initiated in the model organism Saccharomyces cerevisiae. The structure of a late 40S assembly intermediate was determined at 18 Å resolution using cryo-electron microscopy (cryo-EM). The binding sites of the seven AFs bound to this intermediate were identified from cryo-EM structures of particles lacking individual AFs. The positions of AFs as well as the rRNA structure in this pre-40S particle indicate that all seven AFs cooperate to prevent premature translation initiation on multiple levels: by blocking the binding of translation initiation factors, preventing the binding of mRNA, inhibiting joining with the 60S subunit, and preventing formation of the first peptide bond. In vivo data suggest that dissociation of most AFs from this intermediate occurs following regulated but translationally-unproductive joining of 60S subunits. The resulting 80S particles, containing pre-40S and mature 60S subunits, are an obligate intermediate on the 40S maturation pathway and accumulate in the absence of the assembly factor Fap7, an ATPase essential for 40S maturation. Data herein and in the literature suggest that these 80S particles are formed without the help of translation factors that bind to free 40S subunits, as these binding sites are blocked by assembly factors. In contrast, the formation of theses 80S particles requires the translation factors eIF5B and Rli1, which regulate subunit joining and promote dissociation of empty 80S particles, respectively. Furthermore, eIF5A is required for dissociation of the kinase Rio2. To further understand the role of ATP hydrolysis by Fap7 in this process, its ATPase cycle was dissected in vitro showing that ATP binding and hydrolysis lead to a cycle of conformational changes, which include dimerization of the protein in an ATP dependent manner and conformational rearrangement of its C-terminus in response to ATP hydrolysis. Finally, it is shown that Fap7 directly interacts with Dim1 in vitro, further defining its site of interaction with 40S ribosomes.

[发布机构] University of Michigan

[效力级别] Molecular

[关键词] Ribosome Assembly;Molecular;Cellular and Developmental Biology;Science;Chemical Biology