The Effect of Ribosome Assembly Cofactors on In Vitro 30S Subunit Reconstitution

Abstract

Ribosome biogenesis is facilitated by a growing list of assembly cofactors, including helicases, GTPases, chaperones, and other proteins, but the specific functions of many of these assembly cofactors are still unclear. The effect of three assembly cofactors on 30S ribosome assembly was determined in vitro using a previously developed mass spectrometry-based method that monitors the rRNA binding kinetics of ribosomal proteins. The essential GTPase Era caused several late-binding proteins to bind rRNA faster when included in a 30S reconstitution. RimM enabled faster binding of S9 and S19, and inhibited the binding of S12 and S13, perhaps by blocking those proteins' binding sites. RimP caused proteins S5 and S12 to bind dramatically faster. These quantitative kinetic data provide important clues about the roles of these assembly cofactors in the mechanism of 30S biogenesis.

Mutations of ribosomal protein S5 suppress a defect in late-30S ribosomal subunit biogenesis caused by lack of the RbfA biogenesis factor

“…Finding suppressor mutants of S5 is significant since it is the first of the late assembly ribosomal proteins whose binding is promoted by RbfA and RimP (Bunner et al 2010;Sashital et al 2014). It is also significant that S5 binds to a portion of the central pseudoknot (Schuwirth et al 2005), a structure whose assembly depends on RbfA and RimP (Sashital et al 2014).…”

“…The fact that the mutant S5 proteins reverse the lethal effect of overexpressing RimP in the absence of RbfA is consistent with this concept. RimP is known to stabilize the central pseudoknot (Sashital et al 2014) and accelerate binding of S5 and S12 (Bunner et al 2010). The lethality of overexpressing RimP may result from these processes proceeding too quickly, creating kinetically trapped complexes.…”

“…1). All of these factors-Era, RbfA, RimM, and RimP-function late during the cotranscriptional association of r-proteins with the 16S rRNA (Bunner et al 2010;Sashital et al 2014) and RbfA, RimM, and RimP are all implicated in formation and stabilization of the central pseudoknot (Sashital et al 2014). The synthetic lethality from overexpressing RimP in the absence of RbfA is particularly striking, perhaps suggesting that RimP can force bypass of an RbfA "checkpoint" (Connolly and Culver 2013) that coordinates other morphogenetic events with formation of the central pseudoknot.…”

“…Given that the residues altered in S5 are close to the central pseudoknot and that RimP helps form that structure (Sashital et al 2014), it is a reasonable conjecture that the S5 mutant proteins replace RbfA to an extent in modulating RimP activity in forming that structure. However, RimP also accelerates binding of the set of late binding ribosomal proteins (Bunner et al 2010), including S5, which is required for binding of the other proteins. The S5 mutant proteins may, therefore bind especially slowly, thus retarding binding of the later binding proteins, which could preclude the formation of a kinetically trapped complex.…”

“…The complexity of the folding of the rRNAs (Stern et al 1989) and the need to insert ribosomal proteins into this folded structure (Recht and Williamson 2004) present significant thermodynamic barriers to ribosome assembly. Although the precise role of individual factors is frequently unknown, the factors Era, RimM, and RimP have been shown to increase the rate of incorporation of specific r-proteins during in vitro reconstitution of the 30S ribosomal subunits (Bunner et al 2010).…”