Multi-site-specific 16S rRNA methyltransferase RsmF from Thermus thermophilus

Abstract

Cells devote a significant effort toward the production of multiple modified nucleotides in rRNAs, which fine tune the ribosome function. Here, we report that two methyltransferases, RsmB and RsmF, are responsible for all four 5-methylcytidine (m 5 C) modifications in 16S rRNA of Thermus thermophilus. Like Escherichia coli RsmB, T. thermophilus RsmB produces m 5 C967. In contrast to E. coli RsmF, which introduces a single m 5 C1407 modification, T. thermophilus RsmF modifies three positions, generating m 5 C1400 and m 5 C1404 in addition to m 5 C1407. These three residues are clustered near the decoding site of the ribosome, but are situated in distinct structural contexts, suggesting a requirement for flexibility in the RsmF active site that is absent from the E. coli enzyme. Two of these residues, C1400 and C1404, are sufficiently buried in the mature ribosome structure so as to require extensive unfolding of the rRNA to be accessible to RsmF. In vitro, T. thermophilus RsmF methylates C1400, C1404, and C1407 in a 30S subunit substrate, but only C1400 and C1404 when naked 16S rRNA is the substrate. The multispecificity of T. thermophilus RsmF is potentially explained by three crystal structures of the enzyme in a complex with cofactor S-adenosyl-methionine at up to 1.3 Å resolution. In addition to confirming the overall structural similarity to E. coli RsmF, these structures also reveal that key segments in the active site are likely to be dynamic in solution, thereby expanding substrate recognition by T. thermophilus RsmF.

Structure of the human MTERF4–NSUN4 protein complex that regulates mitochondrial ribosome biogenesis

“…NSUN4 contains a typical RNA m5C MTase core domain and lacks variable N-or C-terminal extensions common for RNA recognition in other RNA m5C MTases (18,19,25) (Fig. 1A).…”

“…A search for structural homologs to NSUN4 using Dali (26) revealed significant matches to other RNA m5C MTases (18,19,25), thus enabling a structural-based sequence alignment between NSUN4 and the E. coli structural homologs RsmF and RsmB, as well as RsmF from Thermus thermophilus (TTH) (Fig. S1B).…”

“…2 E-G). The rmsd for the structural superimpositions are 1.9 Å using 243 Cα atoms in RsmF from E. coli, 2.0 Å using 254 Cα atoms in RsmF from TTH, and 2.1 Å using 244 Cα atoms in RsmB from E. coli (18,19,25). In contrast to NSUN4, RsmF and RsmB contain RNA binding domains, which are located in different positions (Fig.…”

“…The crystal structures of the RNA m5C MTases RsmB and RsmF in prokaryotes (18,19) display a conserved MTase domain flanked by variable N-or C-terminal extensions proposed to function as RNA-binding modules. The methyl donor S-adenosyl-L-methionine (SAM) is bound at the base of a positively charged cleft in the MTase domain near two conserved cysteins important for the methylation mechanism.…”

Intrinsic resistance to aminoglycosides in Enterococcus faecium is conferred by the 16S rRNA m5C1404-specific methyltransferase EfmM

“…RsmF is an endogenous housekeeping methyltransferase found in Escherichia coli, and methylates the C5-position of C1407 in the ribosomal A-site without affecting aminoglycoside binding. Distinctive features in the structures of E. faecium EfmM, E. coli RsmF, and the most recently reported Thermus thermophilus RsmF (Demirci et al 2010) indicate the molecular basis for their respective nucleotide specificities.…”