Ribosome Assembly Factors Prevent Premature Translation Initiation by 40 S Assembly Intermediates

Abstract

Ribosome assembly in eukaryotes requires approximately 200 essential assembly factors (AFs), and occurs via ordered events that initiate in the nucleolus and culminate in the cytoplasm. Here we present the cryo-electron microscopy (cryo-EM) structure of a late cytoplasmic 40S ribosome assembly intermediate from Saccharomyces cerevisiae. The positions of bound AFs were defined using cryo-EM reconstructions of pre-ribosomal complexes lacking individual components. All seven AFs are positioned to prevent each step in the translation initiation pathway by obstructing the binding sites for initiation factors, by preventing the opening of the mRNA channel, by blocking 60S subunit joining, and by disrupting the decoding site. We suggest that these highly redundant mechanisms ensure that pre-40S particles do not enter the translation pathway, which would result in their rapid degradation. Implications for the regulation of 40S maturation are also discussed.

Essential ribosome assembly factor Fap7 regulates a hierarchy of RNA–protein interactions during small ribosomal subunit biogenesis

“…Therefore, other ribosome assembly factors and/or rRNA elements in the structural context of the nascent SSU may be required for the timed release and activation of Nob1. Although no ribosome assembly factors interacting with Fap7 have been described, Dim2 (Pno1) and the kinase Rio2 interact directly with Nob1 (11,17) and may modify its structure, activity, and its interaction with Fap7 and RNA. In addition, elongation factor eIF5b and the RNA helicase Prp43 stimulate D-site cleavage by Nob1 in pre-40S particles (14,18).…”

“…These are then further processed and remodeled. Immediately before export into the cytoplasm the pre-SSU contains all but two (Rps10, Rps26) of the ribosomal proteins and seven additional ribosome assembly factors, including the endonuclease Nob1 (11). When Fap7 is depleted in yeast or mutated in its Walker B motif, the nascent SSU lacks a third ribosomal protein, Rps14 (12).…”

A Puzzle of Life: Crafting Ribosomal Subunits

“…Subsequently, the purification of additional preribosomal particles provided a detailed spatiotemporal picture of the preribosomal intermediates and their composition [5,9,14] (Figure 2). A few of these preribosomal particles were visualized at relatively low resolution by EM [21][22][23][24]. Recent progress in cryo-EM has enabled a quantum jump by permitting the visualization of preribosomal particles at atomic or near-atomic resolution [25][26][27][28][29][30][31][32][33].…”

Initial bridges between two ribosomal subunits are formed within 9.4 milliseconds, as studied by time-resolved cryo-EM

“…16S RNA bridges B5 and B6 have intersubunit contacts on the 30S subunit contributed entirely by helix 44. Helix 44 has been shown to be organized late during the assembly of the 30S subunit and has been found to be disordered (18,19) or distorted (20)(21)(22) in structures of some of the assembly intermediates of the small subunit from various species.…”