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Structure of the thiostrepton resistance methyltransferase.S-adenosyl-L-methionine complex and its interaction with ribosomal RNA.

Dunstan MS, Hang PC, Zelinskaya NV, Honek JF, Conn GL - J. Biol. Chem. (2009)

Bottom Line: The x-ray crystal structure of the thiostrepton resistance RNA methyltransferase (Tsr).S-adenosyl-L-methionine (AdoMet) complex was determined at 2.45-A resolution.In vitro methylation assays show that Tsr activity is optimal against a 29-nucleotide hairpin rRNA though the full 58-nucleotide L11-binding domain and intact 23 S rRNA are also effective substrates.Furthermore, a predicted interaction with this internal loop by Tsr amino acid Phe-88 was confirmed by mutagenesis and RNA binding experiments.

View Article: PubMed Central - PubMed

Affiliation: Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.

ABSTRACT
The x-ray crystal structure of the thiostrepton resistance RNA methyltransferase (Tsr).S-adenosyl-L-methionine (AdoMet) complex was determined at 2.45-A resolution. Tsr is definitively confirmed as a Class IV methyltransferase of the SpoU family with an N-terminal "L30-like" putative target recognition domain. The structure and our in vitro analysis of the interaction of Tsr with its target domain from 23 S ribosomal RNA (rRNA) demonstrate that the active biological unit is a Tsr homodimer. In vitro methylation assays show that Tsr activity is optimal against a 29-nucleotide hairpin rRNA though the full 58-nucleotide L11-binding domain and intact 23 S rRNA are also effective substrates. Molecular docking experiments predict that Tsr.rRNA binding is dictated entirely by the sequence and structure of the rRNA hairpin containing the A1067 target nucleotide and is most likely driven primarily by large complementary electrostatic surfaces. One L30-like domain is predicted to bind the target loop and the other is near an internal loop more distant from the target site where a nucleotide change (U1061 to A) also decreases methylation by Tsr. Furthermore, a predicted interaction with this internal loop by Tsr amino acid Phe-88 was confirmed by mutagenesis and RNA binding experiments. We therefore propose that Tsr achieves its absolute target specificity using the N-terminal domains of each monomer in combination to recognize the two distinct structural elements of the target rRNA hairpin such that both Tsr subunits contribute directly to the positioning of the target nucleotide on the enzyme.

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rRNA binding and methylation. A, the 58-nucleotide L11 rRNA binding domain containing the Tsr target nucleotide A1067. Boxed regions correspond to smaller RNA hairpins: 17 nucleotides (solid line) and 29 nucleotides (dashed). A modification of the 3′-UU end for the latter RNA to generate Watson-Crick pairing is indicated. The mutation U1061 to A is indicated, and the internal loop within Helix A is shown in outline type. B, gel electrophoresis shift assays with wild-type 58-nucleotide (upper panel) and 29-nucleotide (lower panel) RNAs at a constant concentration of 6 μm RNA per assay and Tsr input at the concentrations indicated above each gel. Free RNA (▴), RNA-Tsr dimer 1:1 complex (*) and higher molecular weight complexes (**) are indicated on the right hand side. C, gel filtration chromatography of 1:1 mixtures of wild-type (black) and U1061A mutant (gray) 58-nucleotide RNAs (3 μm) and Tsr dimer (3 μm). Elution from the column was monitored at 260 (solid line) and 230 nm (dashed line). The content of each peak is identified as indicated on the basis of apparent molecular weight and relative intensity of absorbance at each wavelength. D, methylation activity was measured for 23 S rRNA and three wild-type (58, 29, and 17 nucleotides) and two U1061A mutant (58 and 29 nucleotides) L11-binding domain RNAs by 3H incorporation. Solid bars represent data at the 10-min time point, and where present open bars represent the 30-min time point (both were measured for all RNAs but for some maximum methylation was reached by the earlier time point). Control experiments used an unrelated 54-nucleotide RNA (see supplemental Fig. S4). Error bars are the standard deviation from at least three independent experiments.
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Figure 2: rRNA binding and methylation. A, the 58-nucleotide L11 rRNA binding domain containing the Tsr target nucleotide A1067. Boxed regions correspond to smaller RNA hairpins: 17 nucleotides (solid line) and 29 nucleotides (dashed). A modification of the 3′-UU end for the latter RNA to generate Watson-Crick pairing is indicated. The mutation U1061 to A is indicated, and the internal loop within Helix A is shown in outline type. B, gel electrophoresis shift assays with wild-type 58-nucleotide (upper panel) and 29-nucleotide (lower panel) RNAs at a constant concentration of 6 μm RNA per assay and Tsr input at the concentrations indicated above each gel. Free RNA (▴), RNA-Tsr dimer 1:1 complex (*) and higher molecular weight complexes (**) are indicated on the right hand side. C, gel filtration chromatography of 1:1 mixtures of wild-type (black) and U1061A mutant (gray) 58-nucleotide RNAs (3 μm) and Tsr dimer (3 μm). Elution from the column was monitored at 260 (solid line) and 230 nm (dashed line). The content of each peak is identified as indicated on the basis of apparent molecular weight and relative intensity of absorbance at each wavelength. D, methylation activity was measured for 23 S rRNA and three wild-type (58, 29, and 17 nucleotides) and two U1061A mutant (58 and 29 nucleotides) L11-binding domain RNAs by 3H incorporation. Solid bars represent data at the 10-min time point, and where present open bars represent the 30-min time point (both were measured for all RNAs but for some maximum methylation was reached by the earlier time point). Control experiments used an unrelated 54-nucleotide RNA (see supplemental Fig. S4). Error bars are the standard deviation from at least three independent experiments.

Mentions: Gel mobility shift assays were used to examine the Tsr-rRNA interaction using both the entire 58-nucleotide L11-binding domain RNA and a 29-nucleotide hairpin containing the A1067 target site (Fig. 2). As expected for a SpoU MTase, we observed a major complex with a 2:1 protein:rRNA stoichiometry for both the 58- and 29-nucleotide rRNA fragments. Higher molecular weight complexes were also seen with increasing protein concentration that have been attributed to tetramer enzyme·RNA complexes for another SpoU MTase (41). These results show that for both RNAs the functional complex is a Tsr dimer bound to one target rRNA. We also examined a 58-nucleotide rRNA containing a U1061 to A mutation (U1061A RNA) within the internal bulge of Helix A, the only site distant from the target loop where mutation dramatically reduces Tsr methylation activity (18). Binding of the U1061A rRNA was significantly weaker with higher molecular weight species dominating at higher Tsr concentrations (supplemental Fig. S3).


Structure of the thiostrepton resistance methyltransferase.S-adenosyl-L-methionine complex and its interaction with ribosomal RNA.

Dunstan MS, Hang PC, Zelinskaya NV, Honek JF, Conn GL - J. Biol. Chem. (2009)

rRNA binding and methylation. A, the 58-nucleotide L11 rRNA binding domain containing the Tsr target nucleotide A1067. Boxed regions correspond to smaller RNA hairpins: 17 nucleotides (solid line) and 29 nucleotides (dashed). A modification of the 3′-UU end for the latter RNA to generate Watson-Crick pairing is indicated. The mutation U1061 to A is indicated, and the internal loop within Helix A is shown in outline type. B, gel electrophoresis shift assays with wild-type 58-nucleotide (upper panel) and 29-nucleotide (lower panel) RNAs at a constant concentration of 6 μm RNA per assay and Tsr input at the concentrations indicated above each gel. Free RNA (▴), RNA-Tsr dimer 1:1 complex (*) and higher molecular weight complexes (**) are indicated on the right hand side. C, gel filtration chromatography of 1:1 mixtures of wild-type (black) and U1061A mutant (gray) 58-nucleotide RNAs (3 μm) and Tsr dimer (3 μm). Elution from the column was monitored at 260 (solid line) and 230 nm (dashed line). The content of each peak is identified as indicated on the basis of apparent molecular weight and relative intensity of absorbance at each wavelength. D, methylation activity was measured for 23 S rRNA and three wild-type (58, 29, and 17 nucleotides) and two U1061A mutant (58 and 29 nucleotides) L11-binding domain RNAs by 3H incorporation. Solid bars represent data at the 10-min time point, and where present open bars represent the 30-min time point (both were measured for all RNAs but for some maximum methylation was reached by the earlier time point). Control experiments used an unrelated 54-nucleotide RNA (see supplemental Fig. S4). Error bars are the standard deviation from at least three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2719339&req=5

Figure 2: rRNA binding and methylation. A, the 58-nucleotide L11 rRNA binding domain containing the Tsr target nucleotide A1067. Boxed regions correspond to smaller RNA hairpins: 17 nucleotides (solid line) and 29 nucleotides (dashed). A modification of the 3′-UU end for the latter RNA to generate Watson-Crick pairing is indicated. The mutation U1061 to A is indicated, and the internal loop within Helix A is shown in outline type. B, gel electrophoresis shift assays with wild-type 58-nucleotide (upper panel) and 29-nucleotide (lower panel) RNAs at a constant concentration of 6 μm RNA per assay and Tsr input at the concentrations indicated above each gel. Free RNA (▴), RNA-Tsr dimer 1:1 complex (*) and higher molecular weight complexes (**) are indicated on the right hand side. C, gel filtration chromatography of 1:1 mixtures of wild-type (black) and U1061A mutant (gray) 58-nucleotide RNAs (3 μm) and Tsr dimer (3 μm). Elution from the column was monitored at 260 (solid line) and 230 nm (dashed line). The content of each peak is identified as indicated on the basis of apparent molecular weight and relative intensity of absorbance at each wavelength. D, methylation activity was measured for 23 S rRNA and three wild-type (58, 29, and 17 nucleotides) and two U1061A mutant (58 and 29 nucleotides) L11-binding domain RNAs by 3H incorporation. Solid bars represent data at the 10-min time point, and where present open bars represent the 30-min time point (both were measured for all RNAs but for some maximum methylation was reached by the earlier time point). Control experiments used an unrelated 54-nucleotide RNA (see supplemental Fig. S4). Error bars are the standard deviation from at least three independent experiments.
Mentions: Gel mobility shift assays were used to examine the Tsr-rRNA interaction using both the entire 58-nucleotide L11-binding domain RNA and a 29-nucleotide hairpin containing the A1067 target site (Fig. 2). As expected for a SpoU MTase, we observed a major complex with a 2:1 protein:rRNA stoichiometry for both the 58- and 29-nucleotide rRNA fragments. Higher molecular weight complexes were also seen with increasing protein concentration that have been attributed to tetramer enzyme·RNA complexes for another SpoU MTase (41). These results show that for both RNAs the functional complex is a Tsr dimer bound to one target rRNA. We also examined a 58-nucleotide rRNA containing a U1061 to A mutation (U1061A RNA) within the internal bulge of Helix A, the only site distant from the target loop where mutation dramatically reduces Tsr methylation activity (18). Binding of the U1061A rRNA was significantly weaker with higher molecular weight species dominating at higher Tsr concentrations (supplemental Fig. S3).

Bottom Line: The x-ray crystal structure of the thiostrepton resistance RNA methyltransferase (Tsr).S-adenosyl-L-methionine (AdoMet) complex was determined at 2.45-A resolution.In vitro methylation assays show that Tsr activity is optimal against a 29-nucleotide hairpin rRNA though the full 58-nucleotide L11-binding domain and intact 23 S rRNA are also effective substrates.Furthermore, a predicted interaction with this internal loop by Tsr amino acid Phe-88 was confirmed by mutagenesis and RNA binding experiments.

View Article: PubMed Central - PubMed

Affiliation: Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.

ABSTRACT
The x-ray crystal structure of the thiostrepton resistance RNA methyltransferase (Tsr).S-adenosyl-L-methionine (AdoMet) complex was determined at 2.45-A resolution. Tsr is definitively confirmed as a Class IV methyltransferase of the SpoU family with an N-terminal "L30-like" putative target recognition domain. The structure and our in vitro analysis of the interaction of Tsr with its target domain from 23 S ribosomal RNA (rRNA) demonstrate that the active biological unit is a Tsr homodimer. In vitro methylation assays show that Tsr activity is optimal against a 29-nucleotide hairpin rRNA though the full 58-nucleotide L11-binding domain and intact 23 S rRNA are also effective substrates. Molecular docking experiments predict that Tsr.rRNA binding is dictated entirely by the sequence and structure of the rRNA hairpin containing the A1067 target nucleotide and is most likely driven primarily by large complementary electrostatic surfaces. One L30-like domain is predicted to bind the target loop and the other is near an internal loop more distant from the target site where a nucleotide change (U1061 to A) also decreases methylation by Tsr. Furthermore, a predicted interaction with this internal loop by Tsr amino acid Phe-88 was confirmed by mutagenesis and RNA binding experiments. We therefore propose that Tsr achieves its absolute target specificity using the N-terminal domains of each monomer in combination to recognize the two distinct structural elements of the target rRNA hairpin such that both Tsr subunits contribute directly to the positioning of the target nucleotide on the enzyme.

Show MeSH
Related in: MedlinePlus