Rare ribosomal RNA sequences from archaea stabilize the bacterial ribosome

Abstract

The ribosome serves as the universally conserved translator of the genetic code into proteins and supports life across diverse temperatures ranging from below freezing to above 120°C. Ribosomes are capable of functioning across this wide range of temperatures even though the catalytic site for peptide bond formation, the peptidyl transferase center, is nearly universally conserved. Here we find that Thermoproteota, a phylum of thermophilic Archaea, substitute cytidine for uridine at large subunit rRNA positions 2554 and 2555 (Escherichia coli numbering) in the A loop, immediately adjacent to the binding site for the 3′-end of A-site tRNA. We show by cryo-EM that E. coli ribosomes with uridine to cytidine mutations at these positions retain the proper fold and post-transcriptional modification of the A loop. Additionally, these mutations do not affect cellular growth, protect the large ribosomal subunit from thermal denaturation, and increase the mutational robustness of nucleotides in the peptidyl transferase center. This work identifies sequence variation across archaeal ribosomes in the peptidyl transferase center that likely confers stabilization of the ribosome at high temperatures and develops a stable mutant bacterial ribosome that can act as a scaffold for future ribosome engineering efforts.

Interactions between terminal ribosomal RNA helices stabilize theEscherichia colilarge ribosomal subunit

“…Mutant E. coli 50S ribosomal subunits were expressed and purified as previously described (14). Briefly, E. coli NEB Express I q cells were transformed with the corresponding pLK35 plasmid.…”

“…tRNA fMet with a C1G mutation and a modified terminal 3′-NH2-ATP was in vitro transcribed and modified as previously described (14). fMet-tRNA fMet was prepared enzymatically as described (18).…”

“…Junk particles were rejected and a second round of 2D classification was performed to further remove the remaining junk particles. Selected particles were re-extracted and Fourier-cropped to 1/4 of the box size and 3D classification was performed with heterogeneous refinement in cryoSPARC using a 70S ribosome map from (14). Classes containing features consistent with 70S ribosomes were combined and subjected to another round of 3D classification.…”

“…We examined the H1-H94-H98 interaction in the cryo-EM structures of a WT 70S ribosome (19) and ribosomes with an MS2-V2 tag (14). In the WT ribosome, there is clear density for G9 (H1), A10 (H1), and U2629 (H94) sugars and bases.…”

“…MS2 tags inserted in H98 of 23S rRNA have been used extensively to enable the investigation of rRNA function (10, 11), in engineered ribosomes (12), and for in vivo ribosome tracking (13). Although E. coli 50S ribosomal subunits with MS2 tags inserted into H98 retain wild-type (WT) in vivo and in vitro function, we previously showed that these 50S subunits have lower thermostability compared to WT 50S subunits (14).…”