Structural dynamics of the yeast Shwachman-Diamond syndrome protein (Sdo1) on the ribosome and its implication in the 60S subunit maturation

The human Shwachman-Diamond syndrome (SDS) is an autosomal recessive disease caused by mutations in a highly conserved ribosome assembly factor SBDS. The functional role of SBDS is to cooperate with another assembly factor, elongation factor 1-like (Efl1), to promote the release of eukaryotic initiation factor 6 (eIF6) from the late-stage cytoplasmic 60S precursors. In the present work, we characterized, both biochemically and structurally, the interaction between the 60S subunit and SBDS protein (Sdo1p) from yeast. Our data show that Sdo1p interacts tightly with the mature 60S subunit in vitro through its domain I and II, and is capable of bridging two 60S subunits to form a stable 2:2 dimer. Structural analysis indicates that Sdo1p bind to the ribosomal P-site, in the proximity of uL16 and uL5, and with direct contact to H69 and H38. The dynamic nature of Sdo1p on the 60S subunit, together with its strategic binding position, suggests a surveillance role of Sdo1p in monitoring the conformational maturation of the ribosomal P-site. Altogether, our data support a conformational signal-relay cascade during late-stage 60S maturation, involving uL16, Sdo1p, and Efl1p, which interrogates the functional P-site to control the departure of the anti-association factor eIF6.

Structural insights into the assembly of the 30S ribosomal subunit in vivo: functional role of S5 and location of the 17S rRNA precursor sequence

The in vivo assembly of ribosomal subunits is a highly complex process, with a tight coordination between protein assembly and rRNA maturation events, such as folding and processing of rRNA precursors, as well as modifications of selected bases. In the cell, a large number of factors are required to ensure the efficiency and fidelity of subunit production. Here we characterize the immature 30S subunits accumulated in a factor-null Escherichia coli strain (∆rsgA∆rbfA). The immature 30S subunits isolated with varying salt concentrations in the buffer system show interesting differences on both protein composition and structure. Specifically, intermediates derived under the two contrasting salt conditions (high and low) likely reflect two distinctive assembly stages, the relatively early and late stages of the 3' domain assembly, respectively. Detailed structural analysis demonstrates a mechanistic coupling between the maturation of the 5' end of the 17S rRNA and the assembly of the 30S head domain, and attributes a unique role of S5 in coordinating these two events. Furthermore, our structural results likely reveal the location of the unprocessed terminal sequences of the 17S rRNA, and suggest that the maturation events of the 17S rRNA could be employed as quality control mechanisms on subunit production and protein translation.