50S subunit recognition and modification by the Mycobacterium tuberculosis ribosomal RNA methyltransferase TlyA

Abstract

Changes in bacterial ribosomal RNA (rRNA) methylation status can alter the activity of diverse groups of ribosome-targeting antibiotics. Typically, such modifications are incorporated by a single methyltransferase that acts on one nucleotide target and rRNA methylation directly prevents drug binding, thereby conferring drug resistance. However, loss of intrinsic methylation can also result in antibiotic resistance. For example, Mycobacterium tuberculosis (Mtb) becomes sensitized to tuberactinomycin antibiotics, such as capreomycin and viomycin, due to the action of the intrinsic methyltransferase TlyA. TlyA is unique among antibiotic resistance-associated methyltransferases as it has dual 16S and 23S rRNA substrate specificity and can incorporate cytidine-2’-O-methylations within two structurally distinct contexts. How TlyA accomplishes this feat of dual-target molecular recognition is currently unknown. Here, we report the structure of the Mtb 50S-TlyA subunit complex trapped in a post-catalytic state with a S-adenosyl-L-methionine analog using single-particle cryogenic electron microscopy. This structure, together with complementary site-directed mutagenesis and methyltransferase functional analyses, reveals critical roles in 23S rRNA substrate recognition for conserved residues across an interaction surface that spans both TlyA domains. These interactions position the TlyA active site over the target nucleotide C2144 which is flipped from 23S Helix 69 in a process stabilized by stacking of TlyA residue Phe157 on the adjacent A2143. This work reveals critical aspects of substrate recognition by TlyA and suggests that base flipping is likely a common strategy among rRNA methyltransferase enzymes even in cases where the target site is accessible without such structural reorganization.

Functional Genomics Identified Novel Genes Involved in Growth at Low Temperatures in Listeria monocytogenes

“…A characteristic feature is the arrangement of α-helices and β-sheets in the FtsJ domain, constituted by 7 β-sheets surrounded by 7 α-helices – a layout very typical for RNA methyltransferases. A similar arrangement occurs in the larger domain of the TlyA protein of Mycobacterium tuberculosis ( 38 ) and YqxC of Bacillus subtilis ( 39 ).…”

Complete list of canonical post-transcriptional modifications in theBacillus subtilisribosome and their link to RbgA driven large subunit assembly

“…Likely, there is no specific hierarchy between RluD h and TlyA h (and to some degree RbgA) as flexibility of the assembly pathway can provide time for the binding and release of these proteins in an arbitrary order. Based on the cryo-EM structures reported previously for RluD and TlyA associated to their substrates (31,34), enzymes access h69 and flip the base out preparing for the catalysis. RluD (PDB ID: subtilis, completion of the modification status strictly follows the order of 23S RNA folding into its canonical conformation.…”

“…tlyA dependent ribose methylation at position 1920-Cm (corresponds to 1949-Cm in B. subtilis) is found in M. tuberculosis. Recently, Laughlin and co-workers demonstrated that recombinant TlyA enzyme stably binds the matured large subunit of M. tuberculosis and efficiently catalyzes cytidine methyl transfer (34).…”

“…The transient nature of rRNA-modification enzyme interactions and low abundance of the assembly intermediates (~ 2% of the total ribosome load in the bacteria cell) in general, make the modification steps difficult to study. Fortuitously, several cryo-EM structures have been reported with modification enzymes bound to their substrates, yet all the findings are currently limited to the late assembly stages (31)(32)(33)(34).…”

“…Quantitative inventory of RNA modifications using isotope ratio MS demonstrates that except for several positions in helix 69, all 23S modifications are fully incorporated into 45S RbgA . The missing modifications are installed by the homologs of pseudouridine synthase RluD and methyltransferases TlyA and RlmH, which are known to be late-acting protein enzymes in other bacteria species (34,36,38). These observations suggest that despite the highly flexible parallel nature of the assembly pathway, modification enzymes elicit their functions along the route strictly cooperating with the folding of the large structural blocks composed of rRNA and r-proteins.…”