Abstract
Dynamic remodelling of intersubunit bridge B2, a conserved RNA domain of the bacterial ribosome connecting helices 44 (h44) and 69 (H69) of the small and large subunit, respectively, impacts translation by controlling intersubunit rotation. Here we show that aminoglycosides chemically related to neomycin—paromomycin, ribostamycin and neamine—each bind to sites within h44 and H69 to perturb bridge B2 and affect subunit rotation. Neomycin and paromomycin, which only differ by their ring-I 6′-polar group, drive subunit rotation in opposite directions. This suggests that their distinct actions hinge on the 6′-substituent and the drug's net positive charge. By solving the crystal structure of the paromomycin–ribosome complex, we observe specific contacts between the apical tip of H69 and the 6′-hydroxyl on paromomycin from within the drug's canonical h44-binding site. These results indicate that aminoglycoside actions must be framed in the context of bridge B2 and their regulation of subunit rotation.
Highlights
Dynamic remodelling of intersubunit bridge B2, a conserved RNA domain of the bacterial ribosome connecting helices 44 (h44) and 69 (H69) of the small and large subunit, respectively, impacts translation by controlling intersubunit rotation
Initiatives spanning the past half century indicate that aminoglycosides with a 2-deoxystreptamine ring target the conserved helix 44 (h44) messenger RNA ‘decoding site’ region of ribosomal RNA within the small (30S) subunit of the bacterial ribosome[7,8], reducing translational fidelity by promoting the promiscuous incorporation of near- and noncognate aminoacyl-transfer RNAs9,10
We present a detailed examination of structure–function relationships in chemically related 4,5-linked aminoglycosides to reveal that neomycin and paromomycin, which differ by only a single functional group, bind h44 and H69 to drive subunit rotation in opposite directions
Summary
Dynamic remodelling of intersubunit bridge B2, a conserved RNA domain of the bacterial ribosome connecting helices 44 (h44) and 69 (H69) of the small and large subunit, respectively, impacts translation by controlling intersubunit rotation. We have shown that neomycin, the most potent and toxic member of the 4,5-linked aminoglycoside class[21] (Fig. 1a), promotes translational miscoding and shutdown by simultaneously binding to the h44-decoding site and the major groove of H69 within the 30S and 50S subunits, respectively[1] (Fig. 1b) These conserved rRNA elements, which comprise the central intersubunit bridge B2, undergo marked structural rearrangements during small-subunit rotation[1], a process underpinning multiple aspects of the translation cycle[19,28,29,30,31]. It is not presently known whether, and to what extent, chemically related 4,5-linked aminoglycosides within the neomycin class impact the dynamics of subunit rotation
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