Base methylations in the double-stranded RNA by a fused methyltransferase bearing unwinding activity

Modifications of rRNAs are clustered in functional regions of the ribosome. In Helix 74 of Escherichia coli 23S rRNA, guanosines at positions 2069 and 2445 are modified to 7-methylguanosine(m(7)G) and N(2)-methylguanosine(m(2)G), respectively. We searched for the gene responsible for m(7)G2069 formation, and identified rlmL, which encodes the methyltransferase for m(2)G2445, as responsible for the biogenesis of m(7)G2069. In vitro methylation of rRNA revealed that rlmL encodes a fused methyltransferase responsible for forming both m(7)G2069 and m(2)G2445. We renamed the gene rlmKL. The N-terminal RlmL activity for m(2)G2445 formation was significantly enhanced by the C-terminal RlmK. Moreover, RlmKL had an unwinding activity of Helix 74, facilitating cooperative methylations of m(7)G2069 and m(2)G2445 during biogenesis of 50S subunit. In fact, we observed that RlmKL was involved in the efficient assembly of 50S subunit in a mutant strain lacking an RNA helicase deaD.     

YebU is a m5C methyltransferase specific for 16 S rRNA nucleotide 1407

The rRNAs in Escherichia coli contain methylations at 24 nucleotides, which collectively are important for ribosome function. Three of these methylations are m5C modifications located at nucleotides C967 and C1407 in 16S rRNA and at nucleotide C1962 in 23S rRNA. Bacterial rRNA modifications generally require specific enzymes, and only one m5C rRNA methyltransferase, RsmB (formerly Fmu) that methylates nucleotide C967, has previously been identified. BLAST searches of the E.coli genome revealed a single gene, yebU, with sufficient similarity to rsmB to encode a putative m5C RNA methyltransferase. This suggested that the yebU gene product modifies C1407 and/or C1962. Here, we analysed the E.coli rRNAs by matrix assisted laser desorption/ionization mass spectrometry and show that inactivation of the yebU gene leads to loss of methylation at C1407 in 16 S rRNA, but does not interfere with methylation at C1962 in 23 S rRNA. Purified recombinant YebU protein retains its specificity for C1407 in vitro, and methylates 30 S subunits (but not naked 16 S rRNA or 70 S ribosomes) isolated from yebU knockout strains. Nucleotide C1407 is located at a functionally active region of the 30 S subunit interface close to the P site, and YebU-directed methylation of this nucleotide seems to be conserved in bacteria. The yebU knockout strains display slower growth and reduced fitness in competition with wild-type cells. We suggest that a more appropriate designation for yebU would be the rRNA small subunit methyltransferase gene rsmF, and that the nomenclature system be extended to include the rRNA methyltransferases that still await identification.     

Methylation at nucleotide G745 or G748 in 23S rRNA distinguishes Gram-negative from Gram-positive bacteria

Bacteria tune the function of their ribosomes by methylating specific rRNA nucleotides. Nucleotide G745 in Escherichia coli 23S rRNA is methylated by the methyltransferase enzyme RrmA, whereas in Streptomyces fradiae, the neighbouring nucleotide G748 is methylated by the enzyme TlrB. Both nucleotides line the peptide exit channel of the ribosome at the binding site of macrolide, lincosamide and streptogramin B antibiotics. Resistance to the macrolide tylosin, which is produced by S. fradiae, is conferred by methylation of G748. RrmA and TlrB are homologues (29% identical), and a database search against all presently available sequences revealed a further two dozen homologues from a wide variety of Bacteria. No homologues were found among the Archaea or Eukarya. The bacterial sequences adhere to the species phylogeny and segregate into two groups, in which the Gram-negative sequences align with RrmA and the Gram-positives with TlrB. Consistently, in more than 20 species tested, the distribution of methylation in the Gram-negative rRNAs (methylated at G745) and the Gram-positives (methylated at G748) perfectly matches the bacterial phylogeny. Cloning and expression of representative methyltransferase genes showed that this specificity of methylation is determined solely by the methyltransferase enzyme and is independent of the origin of the rRNA substrate. This is the first case in which the position of an RNA methylation defines a sharp division between the Gram-negative and Gram-positive bacteria. Given the specificities and distribution of these methyltransferases, we propose a change in the nomenclature of RrmA to RlmAI (rRNA large subunit methyltransferase) and of TlrB to RlmAII.     

The last rRNA methyltransferase of E. coli revealed: The yhiR gene encodes adenine-N6 methyltransferase specific for modification of A2030 of 23S ribosomal RNA

The ribosomal RNA (rRNA) of Escherichia coli contains 24 methylated residues. A set of 22 methyltransferases responsible for modification of 23 residues has been described previously. Herein we report the identification of the yhiR gene as encoding the enzyme that modifies the 23S rRNA nucleotide A2030, the last methylated rRNA nucleotide whose modification enzyme was not known. YhiR prefers protein-free 23S rRNA to ribonucleoprotein particles containing only part of the 50S subunit proteins and does not methylate the assembled 50S subunit. We suggest renaming the yhiR gene to rlmJ according to the rRNA methyltransferase nomenclature. The phenotype of yhiR knockout gene is very mild under various growth conditions and at the stationary phase, except for a small growth advantage at anaerobic conditions. Only minor changes in the total E. coli proteome could be observed in a cell devoid of the 23S rRNA nucleotide A2030 methylation.     

Core sequence in the RNA motif recognized by the ErmE methyltransferase revealed by relaxing the fidelity of the enzyme for its target.

Under physiological conditions, the ErmE methyltransferase specifically modifies a single adenosine within ribosomal RNA (rRNA), and thereby confers resistance to multiple antibiotics. The adenosine (A2058 in Escherichia coli 23S rRNA) lies within a highly conserved structure, and is methylated efficiently, and with equally high fidelity, in rRNAs from phylogenetically diverse bacteria. However, the fidelity of ErmE is reduced when magnesium is removed, and over twenty new sites of ErmE methylation appear in E. coli 16S and 23S rRNAs. These sites show widely different degrees of reactivity to ErmE. The canonical A2058 site is largely unaffected by magnesium depletion and remains the most reactive site in the rRNA. This suggests that methylation at the new sites results from changes in the RNA substrate rather than the methyltransferase. Chemical probing confirms that the rRNA structure opens upon magnesium depletion, exposing potential new interaction sites to the enzyme. The new ErmE sites show homology with the canonical A2058 site, and have the consensus sequence aNNNcgGAHAg (ErmE methylation occurs exclusively at adenosines (underlined); these are preceded by a guanosine, equivalent to G2057; there is a high preference for the adenosine equivalent to A2060; H is any nucleotide except G; N is any nucleotide; and there are slight preferences for the nucleotides shown in lower case). This consensus is believed to represent the core of the motif that Erm methyltransferases recognize at their canonical A2058 site. The data also reveal constraints on the higher order structure of the motif that affect methyltransferase recognition.     

YgdE is the 2'-O-ribose methyltransferase RlmM specific for nucleotide C2498 in bacterial 23S rRNA

The rRNAs of Escherichia coli contain four 2'- O- methylated nucleotides. Similar to other bacterial species and in contrast with Archaea and Eukaryota, the E. coli rRNA modifications are catalysed by specific methyltransferases that find their nucleotide targets without being guided by small complementary RNAs. We show here that the ygdE gene encodes the methyltransferase that catalyses 2'- O- methylation at nucleotide C2498 in the peptidyl transferase loop of E. coli 23S rRNA. Analyses of rRNAs using MALDI mass spectrometry showed that inactivation of the ygdE gene leads to loss of methylation at nucleotide C2498. The loss of ygdE function causes a slight reduction in bacterial fitness. Methylation at C2498 was restored by complementing the knock-out strain with a recombinant copy of ygdE . The recombinant YgdE methyltransferase modifies C2498 in naked 23S rRNA, but not in assembled 50S subunits or ribosomes. Nucleotide C2498 is situated within a highly conserved and heavily modified rRNA sequence, and YgdE's activity is influenced by other modification enzymes that target this region. Phylogenetically, YgdE is placed in the cluster of orthologous groups COG2933 together with S -adenosylmethionine-dependent, Rossmann-fold methyltransferases such as the archaeal and eukaryotic RNA-guided fibrillarins. The ygdE gene has been redesignated rlmM for r RNA l arge subunit m ethyltransferase M .     

A single methyltransferase YefA (RlmCD) catalyses both m5U747 and m5U1939 modifications in Bacillus subtilis 23S rRNA

Methyltransferases that use S-adenosylmethionine (AdoMet) as a cofactor to catalyse 5-methyl uridine (m(5)U) 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 m(5)U modifications. However, as previously shown, the m(5)U54 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 m(5)U-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 m(5)U methyltransferase. To reflect its dual specificity, YefA is renamed RlmCD.     

Ribosomal RNA guanine-(N2)-methyltransferases and their targets

Five nearly universal methylated guanine-(N2) residues are present in bacterial rRNA in the ribosome. To date four out of five ribosomal RNA guanine-(N2)-methyltransferases are described. RsmC(YjjT) methylates G1207 of the 16S rRNA. RlmG(YgjO) and RlmL(YcbY) are responsible for the 23S rRNA m²G1835 and m²G2445 formation, correspondingly. RsmD(YhhF) is necessary for methylation of G966 residue of 16S rRNA. Structure of Escherichia coli RsmD(YhhF) methyltransferase and the structure of the Methanococcus jannaschii RsmC ortholog were determined. All ribosomal guanine-(N2)-methyltransferases have similar AdoMet-binding sites. In relation to the ribosomal substrate recognition, two enzymes that recognize assembled subunits are relatively small single domain proteins and two enzymes that recognize naked rRNA are larger proteins containing separate methyltransferase- and RNA-binding domains. The model for recognition of specific target nucleotide is proposed. The hypothetical role of the m²G residues in rRNA is discussed.     

Identification of the rrmA Gene Encoding the 23S rRNA m1G745 Methyltransferase in Escherichia coli and Characterization of an m1G745-Deficient Mutant

An Escherichia coli mutant lacking the modified nucleotide m¹G in rRNA has previously been isolated (G. R. Björk and L. A. Isaksson, J. Mol. Biol. 51:83–100, 1970). In this study, we localize the position of the m¹G to nucleotide 745 in 23S rRNA and characterize a mutant deficient in this modification. This mutant shows a 40% decreased growth rate in rich media, a drastic reduction in loosely coupled ribosomes, a 20% decreased polypeptide chain elongation rate, and increased resistance to the ribosome binding antibiotic viomycin. The rrmA gene encoding 23S rRNA m¹G745 methyltransferase was mapped to bp 1904000 on the E. coli chromosome and identified to be identical to the previously sequenced gene yebH.     

Characterization of the 23 S ribosomal RNA m5U1939 methyltransferase from Escherichia coli.

An Escherichia coli open reading frame, ygcA, was identified as a putative 23 S ribosomal RNA 5-methyluridine methyltransferase (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762). We have cloned, expressed, and purified the 50-kDa protein encoded by ygcA. The purified enzyme catalyzed the AdoMet-dependent methylation of 23 S rRNA but did not act upon 16 S rRNA or tRNA. A high performance liquid chromatography-based nucleoside analysis identified the reaction product as 5-methyluridine. The enzyme specifically methylated U1939 as determined by a nuclease protection assay and by methylation assays using site-specific mutants of 23 S rRNA. A 40-nucleotide 23 S rRNA fragment (nucleotide 1930--1969) also served as an efficient substrate for the enzyme. The apparent K(m) values for the 40-mer RNA oligonucleotide and AdoMet were 3 and 26 microm, respectively, and the apparent k(cat) was 0.06 s(-1). The enzyme contains two equivalents of iron/monomer and has a sequence motif similar to a motif found in iron-sulfur proteins. We propose to name this gene rumA and accordingly name the protein product as RumA for RNA uridine methyltransferase.     

Methylation of 23S rRNA nucleotide G745 is a secondary function of the RlmAI methyltransferase

Several groups of Gram-negative bacteria possess an RlmA(I) methyltransferase that methylates 23S rRNA nucleotide G745 at the N1 position. Inactivation of rlmA(I) in Acinetobacter calcoaceticus and Escherichia coli reduces growth rates by at least 30%, supposedly due to ribosome malfunction. Wild-type phenotypes are restored by introduction of plasmid-encoded rlmA(I), but not by the orthologous Gram-positive gene rlmA(II) that methylates the neighboring nucleotide G748. Nucleotide G745 interacts with A752 in a manner that does not involve the guanine N1 position. When a cytosine is substituted at A752, a Watson-Crick G745-C752 pair is formed occluding the guanine N1 and greatly reducing RlmA(I) methylation. Methylation is completely abolished by substitution of the G745 base. Intriguingly, the absence of methylation in E. coli rRNA mutant strains causes no reduction in growth rate. Furthermore, the slow-growing rlmA(I) knockout strains of Acinetobacter and E. coli revert to the wild-type growth phenotype after serial passages on agar plates. All the cells tested were pseudorevertants, and none of them had recovered G745 methylation. Analyses of the pseudorevertants failed to reveal second-site mutations in the ribosomal components close to nucleotide G745. The results indicate that cell growth is not dependent on G745 methylation, and that the RlmA(I) methyltransferase therefore has another (as yet unidentified) primary function.     

YccW is the m5C methyltransferase specific for 23S rRNA nucleotide 1962

Methylation at the 5-position of cytosine [m⁵C (5-methylcytidine)] occurs at three RNA nucleotides in Escherichia coli. All these modifications are at highly conserved nucleotides in the rRNAs, and each is catalyzed by its own m⁵C methyltransferase enzyme. Two of the enzymes, RsmB and RsmF, are already known and methylate 16S rRNA at nucleotides C967 and C1407, respectively. Here, we report the identity of the third E. coli m⁵C methyltransferase. Analysis of rRNAs by matrix-assisted laser desorption/ionization mass spectrometry showed that inactivation of the yccW gene leads to loss of m⁵C methylation at nucleotide 1962 in E. coli 23S rRNA. This methylation is restored by complementing the knockout strain with a plasmid-encoded copy of the yccW gene. Purified recombinant YccW protein retains its specificity for C1962 in vitro and methylates naked 23S rRNA isolated from the yccW knockout strain. However, YccW does not methylate assembled 50S subunits, and this is somewhat surprising as the published crystal structures show nucleotide C1962 to be fully accessible at the subunit interface. YccW-directed methylation at nucleotide C1962 is conserved in bacteria, and loss of this methylation in E. coli marginally reduces its growth rate. YccW had previously eluded identification because it displays only limited sequence similarity to the m⁵C methyltransferases RsmB and RsmF and is in fact more similar to known m⁵U (5-methyluridine) RNA methyltransferases. In keeping with the previously proposed nomenclature system for bacterial rRNA methyltransferases, yccW is now designated as the rRNA large subunit methyltransferase gene rlmI.     

Biosynthetic pathway of ribothymidine in B. subtilis and M. lysodeikticus involving different coenzymes for transfer RNA and ribosomal RNA.

Ribothymidine (m5u) in tRNAs of M. lysodeikticus is not derived from methionine. The results indicate that as in tRNAs of B. subtilis a tetrahydrofolate derivative is involved in the formation of m5U, whereas methionine serves as precursor in the biosynthesis of m7G, m1A and m6A. Ribothymidine also occurs in 23S rRNA of B. subtilis and M. lysodeikticus. Approximately 2-3 moles of m5U residues were found per mole of 23S rRNA. In contrast to m5U residues present in tRNAs of B. subtilis and M. lysodeikticus, ribothymidine in 23S rRNA of these organisms and of E. coli is synthesized via S-adenosylmethionine. m6A and m1G, present in E. coli rRNAs, were not detected in rRNAs of (methyl-14C) methionine labeled B. subtilis and M. lysodeikticus.     

Recognition of nucleotide G745 in 23 S ribosomal RNA by the rrmA methyltransferase

Methylation of the N1 position of nucleotide G745 in hairpin 35 of Escherichia coli 23 S ribosomal RNA (rRNA) is mediated by the methyltransferase enzyme RrmA. Lack of G745 methylation results in reduced rates of protein synthesis and growth. Addition of recombinant plasmid-encoded rrmA to an rrmA-deficient strain remedies these defects. Recombinant RrmA was purified and shown to retain its activity and specificity for 23 S rRNA in vitro. The recombinant enzyme was used to define the structures in the rRNA that are necessary for the methyltransferase reaction. Progressive truncation of the rRNA substrate shows that structures in stem-loops 33, 34 and 35 are required for methylation by RrmA. Multiple contacts between nucleotides in these stem-loops and RrmA were confirmed in footprinting experiments. No other RrmA contact was evident elsewhere in the rRNA. The RrmA contact sites on the rRNA are inaccessible in ribosomal particles and, consistent with this, 50 S subunits or 70 S ribosomes are not substrates for RrmA methylation. RrmA resembles the homologous methyltransferase TlrB (specific for nucleotide G748) as well as the Erm methyltransferases (nucleotide A2058), in that all these enzymes methylate their target nucleotides only in the free RNA. After assembly of the 50 S subunit, nucleotides G745, G748 and A2058 come to lie in close proximity lining the peptide exit channel at the site where macrolide, lincosamide and streptogramin B antibiotics bind.     

The 3''-terminal region of bacterial 23S ribosomal RNA: structure and homology with the 3''-terminal region of eukaryotic 28S rRNA and with chloroplast 4.5s rRNA.

The sequence of the 110 nucleotide fragment located at the 3'-end of E.coli, P.vulgaris and A.punctata 23S rRNAs has been determined. The homology between the E.coli and P.vulgaris fragments is 90%, whereas that between the E.coli and A.punctate fragments is only 60%. The three rRNA fragments have sequences compatible with a secondary structure consisting of two hairpins. Using chemical and enzymatic methods recently developed for the study of the secondary structure of RNA, we demonstrated that one of these hairpins and part of the other are actually present in the three 3'-terminal fragments in solution. This supports the existence of these two hairpins in the intact molecule. Indeed, results obtained upon limited digestion of intact 23S RNA with T1 RNase were in good agreement with the existence of these two hairpins. We observed that the primary structures of the 3'-terminal regions of yeast 26S rRNA and X.laevis 28S rRNA are both compatible with a secondary structure similar to that found at the 3'-end of bacterial 23S rRNAs. Furthermore, both tobacco and wheat chloroplast 4.5S rRNAs can also be folded in a similar way as the 3'-terminal region of bacterial 23S rRNA, the 3'-end of chloroplast 4.5S rRNAs being complementary to the 5'-end of chloroplast 23S rRNA. This strongly reinforces the hypothesis that chloroplast 4.5S rRNA originates from the 3'-end of bacterial 23S rRNA and suggests that this rRNA may be base-paired with the 5'-end of chloroplast 23S rRNA. Invariant oligonucleotides are present at identical positions in the homologous secondary structures of E.coli 23S, yeast 26S, X.laevis 28S and wheat and tobacco 4.5S rRNAs. Surprisingly, the sequences of these oligonucleotides are not all conserved in the 3'-terminal regions of A.punctata or even P.vulgaris 23S rRNAs. Results obtained upon mild methylation of E.coli 50S subunits with dimethylsulfate strongly suggest that these invariant oligonucleotides are involved in RNA tertiary structure or in RNA-protein interactions.     

Specificity shifts in the rRNA and tRNA nucleotide targets of archaeal and bacterial m5U methyltransferases

Methyltransferase enzymes that use S-adenosylmethionine as a cofactor to catalyze 5-methyl uridine (m(5)U) formation in tRNAs and rRNAs are widespread in Bacteria and Eukaryota, but are restricted to the Thermococcales and Nanoarchaeota groups amongst the Archaea. The RNA m(5)U methyltransferases appear to have arisen in Bacteria and were then dispersed by horizontal transfer of an rlmD-type gene to the Archaea and Eukaryota. The bacterium Escherichia coli has three gene paralogs and these encode the methyltransferases TrmA that targets m(5)U54 in tRNAs, RlmC (formerly RumB) that modifies m(5)U747 in 23S rRNA, and RlmD (formerly RumA) the archetypical enzyme that is specific for m(5)U1939 in 23S rRNA. The thermococcale archaeon Pyrococcus abyssi possesses two m(5)U methyltransferase paralogs, PAB0719 and PAB0760, with sequences most closely related to the bacterial RlmD. Surprisingly, however, neither of the two P. abyssi enzymes displays RlmD-like activity in vitro. PAB0719 acts in a TrmA-like manner to catalyze m(5)U54 methylation in P. abyssi tRNAs, and here we show that PAB0760 possesses RlmC-like activity and specifically methylates the nucleotide equivalent to U747 in P. abyssi 23S rRNA. The findings indicate that PAB0719 and PAB0760 originated as RlmD-type m(5)U methyltransferases and underwent changes in target specificity after their acquisition by a Thermococcales ancestor from a bacterial source.