A primordial RNA modification enzyme: the case of tRNA (m1A) methyltransferase - PubMed (original) (raw)
A primordial RNA modification enzyme: the case of tRNA (m1A) methyltransferase
Martine Roovers et al. Nucleic Acids Res. 2004.
Abstract
The modified nucleoside 1-methyladenosine (m(1)A) is found in the T-loop of many tRNAs from organisms belonging to the three domains of life (Eukaryota, Bacteria, Archaea). In the T-loop of eukaryotic and bacterial tRNAs, m(1)A is present at position 58, whereas in archaeal tRNAs it is present at position(s) 58 and/or 57, m(1)A57 being the obligatory intermediate in the biosynthesis of 1-methylinosine (m(1)I57). In yeast, the formation of m(1)A58 is catalysed by the essential tRNA (m(1)A58) methyltransferase (MTase), a tetrameric enzyme that is composed of two types of subunits (Gcd14p and Gcd10p), whereas in the bacterium Thermus thermophilus the enzyme is a homotetramer of the TrmI polypeptide. Here, we report that the TrmI enzyme from the archaeon Pyrococcus abyssi is also a homotetramer. However, unlike the bacterial site-specific TrmI MTase, the P.abyssi enzyme is region-specific and catalyses the formation of m(1)A at two adjacent positions (57 and 58) in the T-loop of certain tRNAs. The stabilisation of P.abyssi TrmI at extreme temperatures involves intersubunit disulphide bridges that reinforce the tetrameric oligomerisation, as revealed by biochemical and crystallographic evidences. The origin and evolution of m(1)A MTases is discussed in the context of different hypotheses of the tree of life.
Figures
Figure 1
Affinity-purified P.abyssi PAB0283 protein catalyses the formation of m1A in E.coli tRNA in vitro. (A) SDS–PAGE analysis under reducing conditions of the purified P.abyssi PAB0283 protein. Lane 1, molecular weight marker (Pharmacia-Biotech). Lane 2, 5 µg of purified protein. (B) Autoradiography of a 2D-chromatogram of 5′-phosphate nucleosides on thin layer cellulose plate. Total (bulk) E.coli tRNA (50 µg) was incubated in the presence of [methyl-14C]AdoMet and 5 µg of the purified P.abyssi PAB0283 protein as described in Materials and Methods. After 30 min incubation at 60°C, the tRNA was recovered, digested by nuclease P1 and the resulting nucleotides were analysed by 2D-TLC on a cellulose plate (see Materials and Methods). Circles in dotted lines show the migration of the four canonical nucleotides used as u.v. markers.
Figure 2
Characterisation of the tRNA MTase activity of recombinant PAB0823 protein using different tRNA substrates. Radiolabelled T7 in vitro transcripts of WT T.thermophilus tRNAAsp (A), mutant T.thermophilus tRNAAsp (G57A) (B) and WT yeast tRNAAsp (C) were incubated for 1 h at 60°C in the presence of 20 µg of the purified recombinant PAB0283 protein. After the incubation, the different tRNA transcripts were digested by nuclease P1 or RNAse T2 and the resulting nucleotides were analysed by 2D-TLC on cellulose plates and autoradiography. The nature of the labelled triphosphate nucleoside and the enzyme used to hydrolyse the transcripts are given above each chromatogram. Circles in dotted lines show the migration of the canonical nucleotides used as u.v. markers. m1A>p is for 1-methyladenosine 2′-3′ cyclic phosphate. A schematic representation of the T-loop of the different tRNA substrates is given on the left of each series of chromatograms.
Figure 3
A deaminase present in a crude P.furiosus extract transforms m1A57 preformed in yeast tRNAAsp by the P.abyssi TrmI enzyme into m1I57 and does not deaminate the m1A58 preformed in T.thermophilus tRNAAsp. [α-32P]ATP-labelled yeast tRNAAsp (A and C) and [α-32P]ATP- labelled T.thermophilus tRNAAsp (B and D) were incubated for 1 h at 60°C in the presence of the purified P.abyssi TrmI enzyme and of AdoMet. The transcripts were then recovered and incubated in a crude P.abyssi S30 extract for different periods of time as shown. At the end of the incubation time the transcripts were recovered, digested by nuclease P1 and analysed by 1D-TLC using solvent B (see Materials and Methods) on cellulose plates followed by autoradiography (A and B). (C) and (D) correspond to the autoradiograms of 2D-TLC of the samples incubated for 60 min in the presence of the P.furiosus extract.
Figure 4
Sequence alignment of a few selected tRNA (m1A) MTases from Archaea, Bacteria and Eukaryota (for more extensive alignment with many other tRNA (m1A) MTases, see 30). The size of insertions in S.cerevisiae Gcd14p omitted for clarity is indicated in parentheses. Highly conserved residues are shown on a black background and residues with a similar physico-chemical character are on a grey background. The most conserved motifs among those typical for the RFM superfamily of MTases (51) are indicated.
Figure 5
SDS–PAGE analysis under reducing or non-reducing conditions of the P.abyssi TrmI protein demonstrates the existence of intersubunit disulphide bridges. Before loading, protein samples corresponding to the WT TrmI protein (lanes 1 and 6), C196S mutant (lanes 2 and 7), C233S mutant (lanes 3 and 8) and C196S+C233S double mutant (lanes 4 and 9) were incubated in the presence (lanes 6–9) or absence (lanes 1–4) of 100 mM β-mercaptoethanol for 5 min at 100°C. The molecular mass markers (Pharmacia-Biotech) were loaded into lane 5.
Figure 6
Crystal structure of the catalytic domains (residues 70–250) of the P.abyssi TrmI tetramer. The different monomers are colour-coded as follows: chain A, red; chain B, yellow; chain C, blue; chain D, green. Cysteine residues are shown in CPK representation. (A and B) Two perpendicular ribbon diagrams, showing secondary structure elements. (C) Detailed view of the area delimited in (B) showing disulphide bridges existing between C196 and C233 of different monomers. These disulphide bridges connect C196(C) to C233(A) and C196(D) to C233(B). Another set of symmetry- related disulphide bridges connect C196(A) to C233(C) and C196(B) to C233(D).
Figure 7
Estimation of the apparent molecular mass of the purified WT P.abyssi TrmI protein (A) and of the C196S (B), C233S (C) and C196S+C233S (D) mutants by gel filtration chromatography on a Superdex 200 prep grade 16/60 column (Pharmacia Biotech). The samples consisted of 2.5 mg of purified WT or mutant TrmI proteins in 50 mM Tris–HCl pH 8.5, 500 mM KCl, 200 mM imidazole. Elution was performed with the same buffer. The molecular masses of the proteins were calculated using a standard consisting of carbonic anhydrase from bovine erythrocytes (29 kDa), bovine serum albumin (66 kDa), bovine serum albumin dimer (132 kDa) and β-amylase from sweet potato (200 kDa).
Figure 8
Resistance of the WT P.abyssi TrmI enzyme and of the C196S+C233S mutant to thermodenaturation. The WT (open circles) and mutant (open squares) enzymes (400 µg/ml) were heated for different periods of time at 80 (A) or 85°C (B) in Tris–HCl 50 mM, MgCl2 10 mM. The remaining activity was measured at 70°C using total E.coli tRNA as substrate, [14C-methyl]AdoMet as methyl donor, and 10 µl of the preheated protein solution.
Figure 9
A hypothetical evolutionary scenario of origin and divergence of tRNA (m1A) MTases in the three domains of life. Alternative evolutionary routes (in the prokaryotic part of the tree) have been indicated by broken lines.
References
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