Universal Catalytic Domain Structure of AdoMet-dependent Methyltransferases

Identification of the ribosome binding sites of translation initiation factor IF3 by multidimensional heteronuclear NMR spectroscopy

“…The third region of C-domain implicated in the interaction with ribosomes consists of most of loop L8 and of strand B9, and comprises Thr163, Lys164, Glu166, Arg168, Gln169, Ile171, Val173, Leu174, and Ala175+ All these residues, with the exception of Arg168, which has been identified by site-directed mutagenesis (Petrelli et al+, 1998; R+ Spurio & C+O+ Gualerzi, unpublished results), have been indicated by NMR as being affected by the interaction+ (Figs+ 4 and 5A,B)+ It has been pointed out (Garcia et al+, 1995b) that the structure of the C-domain of IF3 resembles that of the human U1A protein+ The cocrystal structure of this protein complexed with the stem-loop II of U1 snRNA (Oubridge et al+, 1994) and the NMR structure of a complex with another RNA fragment (Allain et al+, 1997) have been determined+ The interaction of U1A with RNA involves strands B1 and B3 containing the consensus RNPs motifs of U1A and two loops: one connecting strand B1 and helix H1 and the other connecting strand B2 and strand B3+ The corresponding regions in IF3 are strands B7 and B9 and the loops L7 and L8 that, as seen above, show interactions with the 30S ribosomes+ Using the DALI Server (Holm & Sander, 1993; http:// www2+ebi+ac+uk/dali), we observed additional structural homology between the IF3 C-domain and the enzymes DNaseI (Lahm & Suck, 1991;Weston et al+, 1992; PDB code: 3dni), HhaI DNA methyltransferase (Klimasauskas et al+, 1994;PDB code: 1hmy), TaqI DNA methyltransferase (Schluckebier et al+, 1995;PDB code: 2adm), the G-domain of the elongation factor G (Czworskowski et al+ 1994; Aevarsson et al+, 1994;PDB code: 1dar), and the N-terminal domain of glutamyl-tRNA synthetase (Nureki et al+, 1995; PDB code: 1gln)+ The similarity in the mode of interaction with nucleic acids make the homologies of IF3 with DNaseI and the HhaI DNA methyltransferase particularly interesting+ For DNaseI (Lahm & Suck, 1991;Weston et al+, 1992) three loops have been implicated in interacting with the minor groove of the DNA+ The first two loops are equivalent to loops L6 and L7 of IF3, while the third loop is not present in IF3+ The residues of the first loop interacting with DNA are Arg9, Glu13, and Thr14 (corresponding to Arg99, Thr102, and Glu104 of IF3), and those of the second loop are Arg41 and Ser43 (corresponding to Phe130 and Ile140 of IF3)+ As seen above, Arg99, Thr102, and Phe130 have been shown to be involved in binding to 30S by several criteria+ The HhaI DNA methyltransferase (Klimasauskas et al+, 1994) consists of a large domain, which is structurally homologous to the C-domain of IF3, and a small domain+ The DNA is bound in the cleft between these two domains+ In the large domain, the loop connecting strand B1 and helix H1 is involved in DNA binding+ This loop, though much longer, corresponds to loop L6 o...…”

Two proteins that form a complex are required for 7-methylguanosine modification of yeast tRNA

“…We employed an assay for the detection of m 7 G modification of tRNA, using S-adenosylmethionine (SAM) as a methyl group donor and [a-32 P]GTP-labeled Saccharomyces cerevisiae intron-containing pre-tRNA Phe as acceptor (Fig+ 1)+ S-adenosylmethionine is used as the methyl donor by most known methyltransferases (Schluckebier et al+, 1995), and pre-tRNA Phe is known to be a substrate for m 7 G formation in vivo and in vitro (Knapp et al+, 1978;Jiang et al+, 1997)+ After incubation of RNA with crude extract and methyl donor, and subsequent P1 nuclease treatment, pG and modified pG derivatives are readily separated by two-dimensional thin layer chromatography (TLC)+ As shown in Figure 2, several modified guanine nucleotides are formed in the presence of crude extract (compare Fig+ 2A, panels a and c)+ We assigned the second fastest migrating spot in the first dimension to m 7 G+ This assignment was based on three criteria: comparison with the published two-dimensional map shown in Figure 2B (Nishimura, 1979a); the observed stimulation of formation of this material by exogenous SAM (Fig+ 2A, panels c and d); and the observation that m 2 2 G formed by purified Trm1 protein (Ellis et al+, 1986) migrates slightly faster in the first dimension than does the spot corresponding to m 7 G (Fig+ 2A, panel e)+ The other guanine nucleotide derivatives, which migrate slower than pm 7 G in the first dimension, correspond to pGp, pm 1 G, and pm 2 G, as indicated in the reference map (Fig+ 2B), and possibly pGm (Jiang et al+, 1997)+ We will provide further evidence below to support our assignment of m 7 G+ As shown in Figure 2C, the separation of m 7 G from other modified guanine nucleotides was easily observed if only the first dimension of the two-dimensional TLC was used+ To identify the yeast gene(s) associated with tRNA m 7 G methyltransferase activity, we screened a genomic set of purified yeast GST-ORF fusion proteins (Martzen et al+, 1999)+ These proteins are derived from a collection of 6,144 yeast strains, each expressing a unique GST-ORF, which are purified in pools after growth of 64 mixtures of 96 strains each+ As shown in Figure 3, m 7 G methyltransferase activity with pre-tRNA Phe was detected in both pools 47 and 51+ Deconvolution of these FIGURE 2. A: Detection of m 7 G methyltransferase activity in yeast extracts+ [a-32 P]-GTP labeled pre-tRNA Phe was incubated with buffer (a, b), 1 mL of 25 mg/mL extract (c, d) or 1 mL of 0+5 mg/mL Trm1 protein (e, f) in the presence of 1 mL SAM (a, c, e) or buffer (b, d, f) for 6 h at 30 8C+ Then samples were treated with P1 nuclease, and modified nucleotides were separated by two-dimensional TLC as described in Materials and Methods+ B: Schematic of expected position of G-modified nucleotides+ The two-dimensional map is adapted from Nishimura (1979a)+ C: One-dimensional chromatography separation of modified guanine nucleotides+ Methyltransferase reactions described in A, separated in the first dimension of the TLC system described above+ FIGURE 3.…”

Structure of the bacteriophage T4 DNA adenine methyltransferase

“…(i) One side of the adenine ring forms edge-to-face van der Waals contacts with the phenyl ring of Phe32 after strand β1; this interaction is highly conserved among DNA MTases 34 . On the other side of the adenine ring lies the side chain of Ile51 (after strand β2).…”