Structural Heterogeneity in Pre-40S Ribosomes

Abstract

SUMMARY Late stage 40S ribosome assembly is a highly regulated, dynamic process that occurs in the cytoplasm, alongside the full translation machinery. Seven assembly factors (AFs) regulate and facilitate maturation, but the mechanisms through which they work remain undetermined. Here, we present a series of structures of the immature small subunit (pre-40S) determined by three-dimensional (3D) cryogenic electron microscopy with 3D sorting to assess the molecule’s heterogeneity. These structures demonstrate extensive structural heterogeneity of interface AFs that likely regulates subunit joining during 40S maturation. We also present structural models for the beak and the platform, two regions where the low resolution of previous studies did not allow for localization of AFs, and the rRNA, respectively. These models are supported by biochemical analyses using point variants and suggest that maturation of the 18S 3’-end is regulated by dissociation of the AF Dim1 from the subunit interface, consistent with previous biochemical analyses.

The ATPase Fap7 Tests the Ability to Carry Out Translocation-like Conformational Changes and Releases Dim1 during 40S Ribosome Maturation

“…No other AFs were found in this analysis (Strunk et al, 2012). This was particularly interesting, as Dim1 is bound near Rps14 on the subunit interface (Johnson et al, 2017; Strunk et al, 2011), raising the possibility that this interaction is functional.…”

“…S7), suggesting that the 40S motions that relocate P-site tRNA to the E-site during translation could release Dim1 during maturation. The 3’-end of 18S rRNA is positioned differently near the endonuclease Nob1 in the presence and absence of Dim1 (Johnson et al, 2017), suggesting that dissociation of Dim1 regulates Nob1 by repositioning pre-18S rRNA. Thus, the Fap7•ATP-dependent release of Dim1 gates 18S rRNA formation, consistent with the previously observed ATP-dependence of 18S rRNA formation, which also requires the presence of Fap7 (Loc’h et al, 2014).…”

“…Furthermore, Fap7 was crosslinked to the AF Dim1 in vivo (Strunk et al, 2012). Dim1 is located on the subunit interface of the 40S (Johnson et al, 2017; Strunk et al, 2011), thereby outlining the Fap7 binding site between Dim1 (near the P-site) and Rps14 (on the platform).…”

A Puzzle of Life: Crafting Ribosomal Subunits

“…Moreover, the release of biogenesis factors, which occupy surfaces that later engage in interactions with translation factors [73,74], must be coordinated with the accommodation of the last r-proteins and the cleavage of the 20S prerRNA [6]. First, formation of the characteristic beak structure involves the stable incorporation of uS3, which is triggered by the phosphorylation-dependent release of Ltv1 [22,75,76] (Figure 2G).…”

“…First, formation of the characteristic beak structure involves the stable incorporation of uS3, which is triggered by the phosphorylation-dependent release of Ltv1 [22,75,76] (Figure 2G). The Rio2 ATPase is strategically positioned between the body and the maturing head in proximity to the decoding center and subsequently may act as a self-releasing checkpoint factor [73,77]. Recruitment of the ATPase Rio1, presumably requiring the prior dissociation of Tsr1 [78], yields late pre-40S ribosomes that are competent to join 60S subunits [79][80][81].…”