Structural and functional studies of the Thermus thermophilus 16S rRNA methyltransferase RsmG

Abstract

The RsmG methyltransferase is responsible for N 7 methylation of G527 of 16S rRNA in bacteria. Here, we report the identification of the Thermus thermophilus rsmG gene, the isolation of rsmG mutants, and the solution of RsmG X-ray crystal structures at up to 1.5 Å resolution. Like their counterparts in other species, T. thermophilus rsmG mutants are weakly resistant to the aminoglycoside antibiotic streptomycin. Growth competition experiments indicate a physiological cost to loss of RsmG activity, consistent with the conservation of the modification site in the decoding region of the ribosome. In contrast to Escherichia coli RsmG, which has been reported to recognize only intact 30S subunits, T. thermophilus RsmG shows no in vitro methylation activity against native 30S subunits, only low activity with 30S subunits at low magnesium concentration, and maximum activity with deproteinized 16S rRNA. Cofactor-bound crystal structures of RsmG reveal a positively charged surface area remote from the active site that binds an adenosine monophosphate molecule. We conclude that an early assembly intermediate is the most likely candidate for the biological substrate of RsmG.

Regulation of expression and catalytic activity of Escherichia coli RsmG methyltransferase

“…In spite of participation of the 530 loop in the decoding process being clear, the precise role of the m 7 G527 modification has remained elusive. Loss of RsmG activity confers low-level streptomycin resistance and inexplicably prompts the appearance of high-level streptomycin resistance mutations in the S12-encoding gene, rpsL (Nishimura et al 2007a,b;Okamoto et al 2007;Gregory et al 2009;Wong et al 2011). Moreover, the effects of rsmG mutations on bacterial fitness and translational accuracy have been reported as being variable, depending on the bacterial species (Nishimura et al 2007b;Okamoto et al 2007;Gregory et al 2009;Ochi et al 2009).…”

“…Loss of RsmG activity confers low-level streptomycin resistance and inexplicably prompts the appearance of high-level streptomycin resistance mutations in the S12-encoding gene, rpsL (Nishimura et al 2007a,b;Okamoto et al 2007;Gregory et al 2009;Wong et al 2011). Moreover, the effects of rsmG mutations on bacterial fitness and translational accuracy have been reported as being variable, depending on the bacterial species (Nishimura et al 2007b;Okamoto et al 2007;Gregory et al 2009;Ochi et al 2009). We recently found that a null rsmG mutation produces an error-prone phenotype in certain genetic backgrounds, which suggests that modification m 7 G527 is important for ribosomal accuracy and that pleitropy may explain, at least partially, the observed variability (A Benítez-Páez, M Villarroya, and M-E Armengod, in prep.…”

“…RsmG belongs to Class I methyltransferases (Schubert et al 2003). Its structure shows a common Rossmann fold where the characteristic AdoMet binding signature motif GxGxG is found at the end of the first b-strand (Romanowski et al 2002;Gregory et al 2009). However, the structural basis for substrate recognition and catalysis by RsmG (as for most m 7 G MTases) remains poorly characterized (Purta et al 2005;Gregory et al 2009;Husain et al 2010).…”

“…Its structure shows a common Rossmann fold where the characteristic AdoMet binding signature motif GxGxG is found at the end of the first b-strand (Romanowski et al 2002;Gregory et al 2009). However, the structural basis for substrate recognition and catalysis by RsmG (as for most m 7 G MTases) remains poorly characterized (Purta et al 2005;Gregory et al 2009;Husain et al 2010). Structures of E. coli and Bacillus subtilis RsmG have been determined in the apo form (Romanowski et al 2002;and PDB id 1XDZ, respectively), whereas RsmG protein from Thermus thermophilus has been crystallized both in the apo form and complexed with AdoMet and AdoHcy (Gregory et al 2009).…”

“…However, the structural basis for substrate recognition and catalysis by RsmG (as for most m 7 G MTases) remains poorly characterized (Purta et al 2005;Gregory et al 2009;Husain et al 2010). Structures of E. coli and Bacillus subtilis RsmG have been determined in the apo form (Romanowski et al 2002;and PDB id 1XDZ, respectively), whereas RsmG protein from Thermus thermophilus has been crystallized both in the apo form and complexed with AdoMet and AdoHcy (Gregory et al 2009). One of the complexes of T. thermophilus RsmG with AdoMet also contained adenosine monophosphate (AMP), suggesting a putative RNA-binding site in the C-terminal region, remote from the active site.…”