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

Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912, USA.
RNA (Impact Factor: 4.94). 08/2010; 16(8):1584-96. DOI: 10.1261/rna.2088310
Source: PubMed


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 A 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.

Download full-text


Available from: Hasan Demirci
  • Source
    • "Notwithstanding, further sequence, structural, and functional characterizations of the known RNA MTases are absolutely essential to: i) clarify the critical amino acids for the function and specificity of MTases; ii) disclose potential new catalytic mechanisms; iii) study the structural rearrangements that some MTases undergo to perform their functions; iv) acquire knowledge of the dual activities of RNA MTases, which are becoming a more frequent event than expected; and v) shed light on the evolutionary origin and relationships among RNA MTases. In recent years, several three-dimensional structures have been solved and some offer insights into catalytic mechanisms of nucleotide methylation [8,35-37]. Similarly, relevant genomic studies have presented important phylogenetic and evolutionary features of RNA MTases [11,38]. Moreover, with the full set of known RNA MTases characterized for the model organism Escherichia coli, new large-scale sequence and genomic studies into the function, variation and diversity of these enzymes responsible for RNA methylation can lead to a better understanding of the origin of this superfamily of enzymes and shed light on both their evolutionarily meaning as well as the link between RNA methylations and bacterial antibiotic resistance. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Background RNA post-transcriptional modification is an exciting field of research that has evidenced this editing process as a sophisticated epigenetic mechanism to fine tune the ribosome function and to control gene expression. Although tRNA modifications seem to be more relevant for the ribosome function and cell physiology as a whole, some rRNA modifications have also been seen to play pivotal roles, essentially those located in central ribosome regions. RNA methylation at nucleobases and ribose moieties of nucleotides appear to frequently modulate its chemistry and structure. RNA methyltransferases comprise a superfamily of highly specialized enzymes that accomplish a wide variety of modifications. These enzymes exhibit a poor degree of sequence similarity in spite of using a common reaction cofactor and modifying the same substrate type. Results Relationships and lineages of RNA methyltransferases have been extensively discussed, but no consensus has been reached. To shed light on this topic, we performed amino acid and codon-based sequence analyses to determine phylogenetic relationships and molecular evolution. We found that most Class I RNA MTases are evolutionarily related to protein and cofactor/vitamin biosynthesis methyltransferases. Additionally, we found that at least nine lineages explain the diversity of RNA MTases. We evidenced that RNA methyltransferases have high content of polar and positively charged amino acid, which coincides with the electrochemistry of their substrates. Conclusions After studying almost 12,000 bacterial genomes and 2,000 patho-pangenomes, we revealed that molecular evolution of Class I methyltransferases matches the different rates of synonymous and non-synonymous substitutions along the coding region. Consequently, evolution on Class I methyltransferases selects against amino acid changes affecting the structure conformation.
    Full-text · Article · Jul 2014 · BMC Research Notes
  • Source
    • "In recent years, several complete modification maps of bacteria, such as Escherichia coli and Thermus thermophilus have been determined, and the modifying enzymes are quite conserved (Ofengand and Del Campo 2004; Guymon et al. 2006; Purta et al. 2009). This suggests that there are common recognition mechanisms and common functional requirements conserved since divergence from their last common ancestor (Demirci et al. 2010). Though many structures of the rRNA methyltransferases (MTases) have been solved, little is known about the process in which they bind the methyl group donor AdoMet and specifically recognize certain nucleotides and then transfer a methyl group to the nucleotide. "
    [Show abstract] [Hide abstract]
    ABSTRACT: RlmG is a specific AdoMet-dependent methyltransferase (MTase) responsible for N²-methylation of G1835 in 23S rRNA of Escherichia coli. Methylation of m²G1835 specifically enhances association of ribosomal subunits and provides a significant advantage for bacteria in osmotic and oxidative stress. Here, the crystal structure of RlmG in complex with AdoMet and its structure in solution were determined. The structure of RlmG is similar to that of the MTase RsmC, consisting of two homologous domains: the N-terminal domain (NTD) in the recognition and binding of the substrate, and the C-terminal domain (CTD) in AdoMet-binding and the catalytic process. However, there are distinct positively charged protuberances and a distribution of conserved residues contributing to the charged surface patch, especially in the NTD of RlmG for direct binding of protein-free rRNA. The RNA-binding properties of the NTD and CTD characterized by both gel electrophoresis mobility shift assays and isothermal titration calorimetry showed that NTD could bind RNA independently and RNA binding was achieved by the NTD, accomplished by a coordinating role of the CTD. The model of the RlmG-AdoMet-RNA complex suggested that RlmG may unfold its substrate RNA in the positively charged cleft between the NTD and CTD, and then G1835 disengages from its Watson-Crick pairing with C1905 and flips out to insert into the active site. Our structure and biochemical studies provide novel insights into the catalytic mechanism of G1835 methylation.
    Preview · Article · Jul 2012 · RNA
  • Source
    • "On the whole, this view has been substantiated by studies of the numerous rRNA modifying enzymes in E. coli [reviewed in (34)] with only a few amendments being necessary. For instance, a handful of enzymes including the pseudouridine synthase RluD (20,36), the highly conserved m6A dimethyltransferase RsmA/KsgA (37–39) and the m5C methyltransferase RsmF of Thermus thermophilus (40) modify multiple sites that are immediately adjacent (RsmA) or within several nucleotides of each other on the bacterial rRNAs (RluD and RsmF). It could be argued that these enzymes engage in a single binding event with the rRNA during which a second or third nucleotide is accommodated into the active site without the need for the enzyme to dissociate from the substrate. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Methyltransferases that use S-adenosylmethionine (AdoMet) as a cofactor to catalyse 5-methyl uridine (m5U) formation in tRNAs and rRNAs are widespread in Bacteria and Eukaryota, and are also found in certain Archaea. These enzymes belong to the COG2265 cluster, and the Gram-negative bacterium Escherichia coli possesses three paralogues. These comprise the methyltransferases TrmA that targets U54 in tRNAs, RlmC that modifies U747 in 23S rRNA and RlmD that is specific for U1939 in 23S rRNA. The tRNAs and rRNAs of the Gram-positive bacterium Bacillus subtilis have the same three m5U modifications. However, as previously shown, the m5U54 modification in B. subtilis tRNAs is catalysed in a fundamentally different manner by the folate-dependent enzyme TrmFO, which is unrelated to the E. coli TrmA. Here, we show that methylation of U747 and U1939 in B. subtilis rRNA is catalysed by a single enzyme, YefA that is a COG2265 member. A recombinant version of YefA functions in an E. coli m5U-null mutant adding the same two rRNA methylations. The findings suggest that during evolution, COG2265 enzymes have undergone a series of changes in target specificity and that YefA is closer to an archetypical m5U methyltransferase. To reflect its dual specificity, YefA is renamed RlmCD.
    Full-text · Article · Aug 2011 · Nucleic Acids Research
Show more