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.

Mutagenesis was utilised to reveal corrole–protein interactions in H-NOX and HasA. The key interaction is a hydrogen bond between the PO unit of the corrole and a protonated histidine residue.

RNA thermosensors (RNATs), found in the 5′ untranslated region (UTR) of some bacterial messenger RNAs (mRNAs), control the translation of the downstream gene in a temperature-dependent manner. In Listeria monocytogenes, the expression of a key transcription factor, PrfA, is mediated by an RNAT in its 5′ UTR. PrfA functions as a master regulator of virulence in L. monocytogenes, controlling the expression of many virulence factors. The temperature-regulated expression of PrfA by its RNAT element serves as a signal of successful host invasion for the bacteria. Structurally, the prfA RNAT bears little resemblance to known families of RNATs, and prior studies demonstrated that the prfA RNAT is highly responsive over a narrow temperature range. Herein, we have undertaken a comprehensive mutational and thermodynamic analysis to ascertain the molecular determinants of temperature sensitivity. We provide evidence to support the idea that the prfA RNAT unfolding is different from that of cssA, a well-characterized RNAT, suggesting that these RNATs function via distinct mechanisms. Our data show that the unfolding of the prfA RNAT occurs in two distinct events and that the internal loops play an important role in mediating the cooperativity of RNAT unfolding. We further demonstrated that regions distal to the ribosome binding site (RBS) not only contribute to RNAT structural stability but also impact translation of the downstream message. Our collective results provide insight connecting the thermal stability of the prfA RNAT structure, unfolding energetics, and translational control.

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.

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.