The cryo-EM structure of hibernating 100S ribosome dimer from pathogenic Staphylococcus aureus

Abstract

Formation of 100S ribosome dimer is generally associated with translation suppression in bacteria. Trans-acting factors ribosome modulation factor (RMF) and hibernating promoting factor (HPF) were shown to directly mediate this process in E. coli. Gram-positive S. aureus lacks an RMF homolog and the structural basis for its 100S formation was not known. Here we report the cryo-electron microscopy structure of the native 100S ribosome from S. aureus, revealing the molecular mechanism of its formation. The structure is distinct from previously reported analogs and relies on the HPF C-terminal extension forming the binding platform for the interactions between both of the small ribosomal subunits. The 100S dimer is formed through interactions between rRNA h26, h40, and protein uS2, involving conformational changes of the head as well as surface regions that could potentially prevent RNA polymerase from docking to the ribosome.

Disassembly of the Staphylococcus aureus hibernating 100S ribosome by an evolutionarily conserved GTPase

“…Furthermore, by preserving a subpool of 100S ribosomes, the ΔhflX mutant becomes more resistant to acute thermal killing (Fig. S5A), which is consistent with our recent observation that a loss of HPF dimerization function sensitizes cells to heat killing (19). More work is certainly needed to better integrate the opposing actions of HPF and HflX into the complex heat stress and stringent response network.…”

“…Furthermore, the parallel beta-sheet interactions between the two 70S ribosomes are not rigid (19), and disrupting the flexible loop linking the CTD and NTD impairs dimerization (18). These findings suggest that the dissociation of 100S ribosome involves an active mechanism to dislodge the dimerizing factors from the ribosome.…”

“…A higher GTP concentration could partially offset the (55). The N-terminal domain of HPF binds to the decoding center of the 30S subunit and inhibits translation, whereas the C-terminal domain (CTD) tethers the two 70S monomers via direct interaction of the HPF-CTD dimer to form the 100S complex (19). The production of (p)ppGpp strongly inhibits the synthesis of hpf and hflX under heat stress.…”

“…It is possible that the "masked" 30S-30S interfaces in the dimer are more resistant to nucleolytic attack, although these regions are not known to be the targets of RNases (58)(59)(60). Perhaps the conformational changes in the 30S subunits (19) reduce the accessibility to RNases, and moreover, translational attenuation may alter the expression of specific factors involved in the ribosome decay pathway. Alternatively, dimerization may simply avoid spurious interactions of RNA polymerase (RNAP) with the inactive 70S ribosome because the dimerization interface, e.g., uS2, overlaps with the RNAP-ribosome contact sites (61).…”

“…1). In addition, the N-terminal domain (NTD) of HPF inhibits translation by sterically occluding the mRNA and the anticodon tRNA binding sites (18)(19)(20). During HPF-induced dimerization, a 30S head rotation of the S. aureus 100S ribosome stabilizes the dimerization interface consisting of the rRNA h26, and h40 and the ribosomal protein uS2 (19).…”