Interactions between terminal ribosomal RNA helices stabilize theE. colilarge ribosomal subunit

Abstract

The ribosome is a large ribonucleoprotein assembly that uses diverse and complex molecular interactions to maintain proper folding.In vivoassembled ribosomes have been isolated using MS2 tags installed in either the 16S or 23S ribosomal RNAs (rRNAs), to enable studies of ribosome structure and functionin vitro. RNA tags in theEscherichia colilarge ribosomal (50S) subunit have commonly been inserted into an extended helix H98 in 23S rRNA, as this addition does not affect cellular growth orin vitroribosome activity. Here, we find thatE. coli50S subunits with MS2 tags inserted in H98 are destabilized compared to wild type (WT) 50S subunits. We identify the loss of RNA-RNA tertiary contacts that bridge helices H1, H94, and H98 as the cause of destabilization. Using cryogenic electron microscopy (cryo-EM), we show that this interaction is disrupted by the addition of the MS2 tag and can be restored through the insertion of a single adenosine in the extended H98 helix. This work establishes ways to improve MS2 tags in the 50S subunit that maintain ribosome stability and investigates a complex RNA tertiary structure that may be important for stability in various bacterial ribosomes.

Domain consolidation in Bacterial 50S assembly revealed by Anti-Sense Oligonucleotide Probing

“…24 In a matured LSU, H1 contacts with domain VI through H94 and H98 and stabilize the LSU. 25 From previous observations, compared to RA20-B-a1, in deaD-B-a1, the H1 formation was observed without other parts in domain VI formed ( Extended Data Fig.7 ab ). Also, it is clear that the docking of domain III does not need domain I with observation of deaD-preB2 ( Extended Data Fig.7 bc ).…”

Ribosome Pool Engineering Increases Protein Biosynthesis Yields

“…While most of the polymorphisms sit on the outer regions of the rRNA and are thus less likely to be directly interacting with translocation processes of tRNAs through the ribosomal active site, some exist in motifs that are known to play dynamic roles in translation activity. For example, the 23S rRNA sequences of operons A, C, E, and H differ from that of B, D, and G at Helix 98 (H98), a motif that has been shown to participate in a tertiary interaction that is important for ribosome stabilization . Similarly, H68, a 23S rRNA helix that is extended by a Watson–Crick (WC) base pair in the A operon is known to be actively involved in dynamic ribosome movements that are necessary for the process of elongation via coordination with the L1 stalk and tRNAs .…”

“…For example, the 23S rRNA sequences of operons A, C, E, and H differ from that of B, D, and G at Helix 98 (H98), a motif that has been shown to participate in a tertiary interaction that is important for ribosome stabilization. 29 Similarly, H68, a 23S rRNA helix that is extended by a Watson−Crick (WC) base pair in the A operon is known to be actively involved in dynamic ribosome movements that are necessary for the process of elongation via coordination with the L1 stalk and tRNAs. 30 The additional WC base pair, which would increase the length of H68, potentially affects the dynamics of the interaction with the L1 stalk, altering the efficiency of the elongation process.…”