The cryo-EM structure of YjeQ bound to the 30S subunit suggests a fidelity checkpoint function for this protein in ribosome assembly

Abstract

Recent work suggests that bacterial YjeQ (RsgA) participates in the late stages of assembly of the 30S subunit and aids the assembly of the decoding center but also binds the mature 30S subunit with high affinity. To determine the function and mechanisms of YjeQ in the context of the mature subunit, we determined the cryo-EM structure of the fully assembled 30S subunit in complex with YjeQ at 5.8-Å resolution. We found that binding of YjeQ stabilizes helix 44 into a conformation similar to that adopted by the subunit during proofreading. This finding indicates that, along with acting as an assembly factor, YjeQ has a role as a checkpoint protein, consisting of testing the proofreading ability of the 30S subunit. The structure also informs the mechanism by which YjeQ implements the release from the 30S subunit of a second assembly factor, called RbfA. Finally, it reveals how the 30S subunit stimulates YjeQ GTPase activity and leads to release of the protein. Checkpoint functions have been described for eukaryotic ribosome assembly factors; however, this work describes an example of a bacterial assembly factor that tests a specific translation mechanism of the 30S subunit.

Structural and functional investigation of the Small Ribosomal Subunit Biogenesis GTPase A (RsgA) from Pseudomonas aeruginosa

“…In this light the functional state of the protein ('on-state'/GTP-bound or 'offstate'/GDP bound) is likely to reflect the metabolic state of the cell. Growing evidences are emerging, delineating the functions of this protein by innovative, structurally informative techniques, such as the recent high-resolution cryo-EM structures of the ribosomal particles [9,10]. Nevertheless many aspects remain to be unveiled and further studies are necessary to address some unresolved key issues.…”

“…RsgA (Small Ribosomal Subunit Biogenesis GTPase A, also known as YjeQ, YloQ or CpgA) is a multidomain GTPase largely conserved in bacteria involved in the last stages of the 30S subunit maturation . The role played by RsgA in ribogenesis has been documented by in vivo experimental evidences showing that strains lacking the rsgA gene (Δ rsgA ) exhibit a decrease in the mature 70S ribosome levels and an accumulation of 30S and 50S subunits as well the accumulation of the immature 17S rRNA .…”

“…Specific nonribosomal proteins, called ribosome assembly factors, such as GTPases [3], ribonucleases, rRNA (and r-protein)-modifying enzymes and helicases assist this process by transient binding to the ribosomal subunits during their assembly [1,4] and preventing the association of incomplete or misfolded ribosomal subunits into the 70S ribosome [2]. RsgA (Small Ribosomal Subunit Biogenesis GTPase A, also known as YjeQ, YloQ or CpgA) is a multidomain GTPase largely conserved in bacteria [5] involved in the last stages of the 30S subunit maturation [3,[6][7][8][9][10]. The role played by RsgA in ribogenesis has been documented by in vivo experimental evidences showing that strains lacking the rsgA gene (DrsgA) exhibit a decrease in the mature 70S ribosome levels and an accumulation of 30S and 50S subunits [8,11,12] as well the accumulation of the immature 17S rRNA [8].…”

IF3 licenses newly made 30S subunits for translation during stress

“…By contrast with IF3, RsgA uses GTP hydrolysis to induce conformational changes that promote the release of RbfA (Goto et al, 2011;Guo et al, 2011;Pedro Lopez-Alonso et al, 2017;Razi et al, 2017). Although this likely requires folding and docking of 16S H44 and H45, we cannot exclude the possibility that RsgA and IF3 displace RbfA by different mechanisms.…”

“…In current models, GTP hydrolysis induces a conformational change within RsgA that promotes the release of RbfA and RsgA. Dissociation of RbfA and RsgA allows 16S helices H44 and H45 to dock with the 30S platform, making the 30S subunit suitable for translation (Goto et al, 2011;Guo et al, 2011;Pedro Lopez-Alonso et al, 2017;Razi et al, 2017).…”