The yfhQ gene of Escherichia coli encodes a tRNA:Cm32/Um32 methyltransferase
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Naturally occurring tRNAs contain numerous modified nucleosides. They are formed by enzymatic modification of the primary transcripts during the complex RNA maturation process. In model organisms Escherichia coli and Saccharomyces cerevisiae most enzymes involved in this process have been identified. Interestingly, it was found that tRNA methylation, one of the most common modifications, can be introduced by S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases (MTases) that belong to two structurally and phylogenetically unrelated protein superfamilies: RFM and SPOUT.
As a part of a large-scale project aiming at characterization of a complete set of RNA modification enzymes of model organisms, we have studied the Escherichia coli proteins YibK, LasT, YfhQ, and YbeA for their ability to introduce the last unassigned methylations of ribose at positions 32 and 34 of the tRNA anticodon loop. We found that YfhQ catalyzes the AdoMet-dependent formation of Cm32 or Um32 in tRNASer1 and tRNAGln2 and that an E. coli strain with a disrupted yfhQ gene lacks the tRNA:Cm32/Um32 methyltransferase activity. Thus, we propose to rename YfhQ as TrMet(Xm32) according to the recently proposed, uniform nomenclature for all RNA modification enzymes, or TrmJ, according to the traditional nomenclature for bacterial tRNA MTases.
Our results reveal that methylation at position 32 is carried out by completely unrelated TrMet(Xm32) enzymes in eukaryota and prokaryota (RFM superfamily member Trm7 and SPOUT superfamily member TrmJ, respectively), mirroring the scenario observed in the case of the m1G37 modification (introduced by the RFM member Trm5 in eukaryota and archaea, and by the SPOUT member TrmD in bacteria).
KeywordsAdoMet Secondary Structure Content Strain MC1061 Anticodon Loop Modify Nucleoside
Ribose methylation is one of the most common modifications. In E. coli it was found to be introduced at different positions of different RNAs by site-specific enzymes, S-adenosyl-L-methionine (AdoMet)-dependent MTases that belong to two structurally and phylogenetically unrelated protein superfamilies: Rossmann-fold MTase (RFM) [7, 8] and SPOUT . These superfamilies have been defined based on sequence and structural comparisons (review: ). The RFM superfamily can be exemplified by the 23S rRNA:Um2552 MTase RrmJ (which is also able to methylate tRNA in vitro, albeit at an unspecified position) [11, 12], while the SPOUT superfamily can be exemplified by the tRNA:Gm18 MTase TrmH [13, 14].
Previously, we found that in S. cerevisiae methylations at positions 32 and 34 are introduced by the Trm7 enzyme, a close homolog of RrmJ and a member of the RFM superfamily . However, apart from the rRNA MTase RrmJ, there are no close homologs of Trm7 in E. coli that could carry out the corresponding reactions in tRNAs , which suggests that modifications at positions 32 and/or 34 in prokaryotic and eukaryotic tRNAs could be carried out by analogous, i.e. unrelated proteins. To test this hypothesis, we assayed the so far functionally uncharacterized members of the SPOUT superfamily : YibK, LasT, YfhQ, and YbeA for their ability to exert methylation of tRNA at positions 32 and/or 34.
Results and discussion
Preparation of the substrates and methyltransferase candidates
Analysis of the tRNA sequences and modified nucleosides in the MODOMICS database  revealed the presence of 2'-O-methylated uridine (Um) in position 32 of the anticodon loop in tRNAGln1 (UUG) and tRNAGln2 (CUG), and 2'-O-methylated cytidine (Cm) at the same position in tRNAfMet1 (CAU), tRNAfMet2 (CAU), tRNASer1 (UGA), and tRNATrp1 (CCA). On the other hand, the nucleoside in position 34 was found to be 2'-O-methylated in tRNALeu4 (UAA) to 5-carboxymethylaminomethyl-2-O-methyluridine (cmnm5Um), and in tRNALeu5 (CAA) to Cm. We selected tRNASer1 and tRNAGln2 as representative substrates for the methylation at position 32, while tRNALeu5 was selected as a representative substrate for the methylation at position 34. Transcripts were generated in vitro using T7-RNA polymerase (see Methods).
yfhQ encodes a MTase responsible for the formation of Cm/Um 32 in the anticodon loop of tRNA
Sequence analysis and modeling of YfhQ reveals a conserved active site common to ribose 2'-O-MTases from the SPOUT superfamily and underscores the convergent evolution with the RFM superfamily
Our results reveal that methylation at position 32 in the anticodon loop of bacterial and eukaryotic tRNAs is carried out by completely unrelated enzymes: a SPOUT superfamily member YfhQ and RFM superfamily member Trm7. This scenario is strikingly similar to the one observed in the case of m1G37 modification, which is carried out by the SPOUT superfamily member TrmD in bacteria and by the RFM superfamily member Trm5 in eukaryota and archaea [31, 32]. On the other hand, methylation of ribose at position 18 is carried out by members of the SPOUT superfamily both in prokaryota and eukaryota: TrmH , and Trm3 , respectively, while the N1-methylation of adenosine A58 (not observed in E. coli, but e.g. in Thermus thermophilus) is catalyzed by members of the RFM superfamily [34, 35]. It is unclear which of these two MTase superfamilies was more ancient and how they replaced each other for the methylation of different positions in tRNAs from different phylogenetic lineages. Among the four members of the SPOUT superfamily studied in this work (YfhQ, YibK, LasT, and YbeA) we were unable to identify the MTase specific for the position 34 in E. coli tRNAs. It remains to be determined if this last missing tRNA MTase is present among the remaining, so far uncharacterized members of SPOUT and RFM superfamilies [8, 9] and whether it uses a known active site or invented a new one for the same reaction. Definitely, more work is needed to elucidate the complicated pathways of evolution of RNA modification systems in all Domains of Life.
Preparation of the substrates
Plasmids containing the yfhQ, yibK, lasT, and ybeA genes inserted into the pCA24N vector and the corresponding E. coli K.O. strains were constructed as described earlier [21, 22]. pBlueScript II KS (+) (Stratagene) has been modified by site-directed mutagenesis to introduce BpiI and Mph1103I cloning sites in pBlueScript II KS (+) polylinker, resulting in pKS_RNA vector. The serT and leuZ genes, encoding tRNASer1 and tRNALeu5 respectively, were PCR-amplified from the E. coli genomic DNA using primers 5'-GCATGCATTGGCGGAAGCGCAGAGATTCGAAC-3' and 5'-GAAGACCCTATAGG AAGTGTGGCCGAGCGGTTG-3' for serT and 5'-TAATGCATGGTACCCGGAG CGGGAC-3' and 5'-AGACCCTATAGCCCGGATGGTGGAATCGGTAG-3' for leuZ. The final PCR product was cloned into the pKS_RNA vector, generating plasmids pKS_SerT and pKS_LeuZ. Transcripts were generated in vitro using T7-RNA polymerase and Mph1103I-cleaved pKS_SerT or pKS_LeuZ plasmids as templates. The tRNAGln2 transcript was generated exactly as described in . Full-length transcripts were purified by 10% polyacrylamide gel electrophoresis.
Expression and purification of the YfhQ, YibK, LasT, and YbeA recombinant proteins
Proteins were expressed in E. coli strain BL21 (DE3). Transformed cells were grown at 37°C in Luria broth (supplemented with chloramphenicol at 30 μg/mL) to an optical density at 660 nm (OD660) of 0.7. At this stage, IPTG (isopropylthiogalactopyranoside) was added up to a final concentration of 1 mM to induce recombinant protein expression. Cells were harvested after 3 hours incubation at 37°C and resuspended in buffer A (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10% glycerol) and lysed by sonication. The lysate was cleared by centrifugation (20,000 × g during 10 min) and was applied to a column of Chelating Sepharose Fast Flow (Pharmacia Biotech) charged with Ni2+. The column was washed with buffer A supplemented with 5 mM imidazole and the adsorbed material was eluted with a linear gradient (0.05 M up to 0.4 M) of imidazole. Eluted fractions were analyzed by SDS-PAGE in the presence of 5–7.5% β-mercaptoethanol. In the higher concentration of β-mercaptoethanol only the monomeric form was observed (Figure 2), while the lower concentrations allowed to observe both the monomeric and dimeric forms. YfhQ was further purified by gel filtration chromatography. The partially purified enzyme was applied on a Superdex 200 column (Pharmacia Biotech) equilibrated with buffer B (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 10% glycerol).
Circular Dichroism analysis
CD spectra were collected on the Jasco-810 spectropolarimeter with a temperature controller. The concentration of YfhQ protein was 5 μM and YibK protein 6.6 μM. Scans were collected at 20°C from 200 to 260, in 1 nm steps, using a 1 mm pathlength cuvette. Secondary structure content was estimated from CD spectrum using the CDpro software .
We thank Catherine Tricot (Institut de Recherches Microbiologiques Wiame, Bruxelles, Belgium) for the help in gel filtration experiments. The work in Poland was supported by the EMBO/HHMI Young Investigator Programme award to J.M.B. The work in Belgium was supported by the Fonds pour la Recherche Fondamentale Collective (FRFC). E.P. was supported by the fellowship from the Commissariat Général aux Relations internationales de la Communauté française de Belgique and by the Centre of Excellence in Molecular BioMedicine project within the 5th Framework Programme of the European Commission (contract QLK6-CT-2002-90363).
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