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Conserved amino acids in each subunit of the heteroligomeric tRNA m1A58 Mtase from Saccharomyces cerevisiae contribute to tRNA binding.

Ozanick SG, Bujnicki JM, Sem DS, Anderson JT - Nucleic Acids Res. (2007)

Bottom Line: Yeast strains expressing trm6 and trm61 mutants exhibited growth phenotypes indicative of reduced m1A formation.In addition, recombinant mutant enzymes had reduced in vitro Mtase activity.We demonstrate that the mutations introduced do not prevent heteroligomer formation and do not disrupt binding of the cofactor S-adenosyl-L-methionine.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Marquette University, P.O. Box 1881, Milwaukee, WI 53201, USA.

ABSTRACT
In Saccharomyces cerevisiae, a two-subunit methyltransferase (Mtase) encoded by the essential genes TRM6 and TRM61 is responsible for the formation of 1-methyladenosine, a modified nucleoside found at position 58 in tRNA that is critical for the stability of tRNA(Met)i The crystal structure of the homotetrameric m1A58 tRNA Mtase from Mycobacterium tuberculosis, TrmI, has been solved and was used as a template to build a model of the yeast m1A58 tRNA Mtase heterotetramer. We altered amino acids in TRM6 and TRM61 that were predicted to be important for the stability of the heteroligomer based on this model. Yeast strains expressing trm6 and trm61 mutants exhibited growth phenotypes indicative of reduced m1A formation. In addition, recombinant mutant enzymes had reduced in vitro Mtase activity. We demonstrate that the mutations introduced do not prevent heteroligomer formation and do not disrupt binding of the cofactor S-adenosyl-L-methionine. Instead, amino acid substitutions in either Trm6p or Trm61p destroy the ability of the yeast m1A58 tRNA Mtase to bind tRNA(Met)i, indicating that each subunit contributes to tRNA binding and suggesting a structural alteration of the substrate-binding pocket occurs when these mutations are present.

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Purification and characterization of recombinant Trm6p/Trm61p complexes. (A) Wild-type and mutant enzymes were purified from E. coli whole cell extract via a polyhistidine tag on Trm6p. Samples were analyzed by SDS-PAGE and stained with Coomassie blue. (B) Fifteen nanomolar of purified recombinant enzyme was incubated with 30 μM 3H-AdoMet and 150 nM in vitro transcribed  (standard reaction), or 150 nM enzyme was incubated with 30 μM 3H-AdoMet and 1.5 μM  (10 × reaction). After precipitation with 5% trichloroacetic acid, insoluble material was collected on nitrocellulose filters and 3H was measured using liquid scintillation. The data reported is the average of duplicate experiments.
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Figure 4: Purification and characterization of recombinant Trm6p/Trm61p complexes. (A) Wild-type and mutant enzymes were purified from E. coli whole cell extract via a polyhistidine tag on Trm6p. Samples were analyzed by SDS-PAGE and stained with Coomassie blue. (B) Fifteen nanomolar of purified recombinant enzyme was incubated with 30 μM 3H-AdoMet and 150 nM in vitro transcribed (standard reaction), or 150 nM enzyme was incubated with 30 μM 3H-AdoMet and 1.5 μM (10 × reaction). After precipitation with 5% trichloroacetic acid, insoluble material was collected on nitrocellulose filters and 3H was measured using liquid scintillation. The data reported is the average of duplicate experiments.

Mentions: In order to produce large quantities of mutant enzymes and be able to combine mutations in both Trm6p and Trm61p, we reconstructed each trm6 mutant in a vector that allows co-expression of TRM6 and TRM61 in bacteria. TRM61 was expressed in E. coli together with either TRM6 (B329), trm6-416 (B332), trm6-420 (B343) or trm6-504 (B333). Using a polyhistidine tag on Trm6p, protein was purified from soluble E. coli extract using affinity chromatography. Trm61p co-purified with both wild-type Trm6p and Trm6-416p, Trm6-420p and Trm6-504p in apparent stoichiometric amounts (Figure 4A). Purified recombinant enzymes were incubated with S-adenosyl-l-[methyl-3H]methionine (3H-AdoMet) and in vitro transcribed . The incorporation of 3H into was monitored by liquid scintillation counting. Under optimal conditions, the wild-type enzyme has been found to convert a maximum of 50% of tRNA substrate to product. In the assays reported in this study, for which the results are shown as the counts per minute of 3H detected, the wild-type enzyme converted ∼40% of substrate to product, but the mutant enzymes lacked Mtase activity (Figure 4B). Since the trm61-3 and trm61-255 mutants were not able to complement a trm61-2 strain, we also reconstructed these mutants and co-expressed them in bacteria with TRM6 (B360 and B428, respectively). In addition, we created a mutant, called trm6-416/trm61-255 (B429), which has the three mutations in trm6-416 combined with the three mutations in trm61-255. All of these enzymes also lacked in vitro Mtase activity (Figure 4B).Figure 4.


Conserved amino acids in each subunit of the heteroligomeric tRNA m1A58 Mtase from Saccharomyces cerevisiae contribute to tRNA binding.

Ozanick SG, Bujnicki JM, Sem DS, Anderson JT - Nucleic Acids Res. (2007)

Purification and characterization of recombinant Trm6p/Trm61p complexes. (A) Wild-type and mutant enzymes were purified from E. coli whole cell extract via a polyhistidine tag on Trm6p. Samples were analyzed by SDS-PAGE and stained with Coomassie blue. (B) Fifteen nanomolar of purified recombinant enzyme was incubated with 30 μM 3H-AdoMet and 150 nM in vitro transcribed  (standard reaction), or 150 nM enzyme was incubated with 30 μM 3H-AdoMet and 1.5 μM  (10 × reaction). After precipitation with 5% trichloroacetic acid, insoluble material was collected on nitrocellulose filters and 3H was measured using liquid scintillation. The data reported is the average of duplicate experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175304&req=5

Figure 4: Purification and characterization of recombinant Trm6p/Trm61p complexes. (A) Wild-type and mutant enzymes were purified from E. coli whole cell extract via a polyhistidine tag on Trm6p. Samples were analyzed by SDS-PAGE and stained with Coomassie blue. (B) Fifteen nanomolar of purified recombinant enzyme was incubated with 30 μM 3H-AdoMet and 150 nM in vitro transcribed (standard reaction), or 150 nM enzyme was incubated with 30 μM 3H-AdoMet and 1.5 μM (10 × reaction). After precipitation with 5% trichloroacetic acid, insoluble material was collected on nitrocellulose filters and 3H was measured using liquid scintillation. The data reported is the average of duplicate experiments.
Mentions: In order to produce large quantities of mutant enzymes and be able to combine mutations in both Trm6p and Trm61p, we reconstructed each trm6 mutant in a vector that allows co-expression of TRM6 and TRM61 in bacteria. TRM61 was expressed in E. coli together with either TRM6 (B329), trm6-416 (B332), trm6-420 (B343) or trm6-504 (B333). Using a polyhistidine tag on Trm6p, protein was purified from soluble E. coli extract using affinity chromatography. Trm61p co-purified with both wild-type Trm6p and Trm6-416p, Trm6-420p and Trm6-504p in apparent stoichiometric amounts (Figure 4A). Purified recombinant enzymes were incubated with S-adenosyl-l-[methyl-3H]methionine (3H-AdoMet) and in vitro transcribed . The incorporation of 3H into was monitored by liquid scintillation counting. Under optimal conditions, the wild-type enzyme has been found to convert a maximum of 50% of tRNA substrate to product. In the assays reported in this study, for which the results are shown as the counts per minute of 3H detected, the wild-type enzyme converted ∼40% of substrate to product, but the mutant enzymes lacked Mtase activity (Figure 4B). Since the trm61-3 and trm61-255 mutants were not able to complement a trm61-2 strain, we also reconstructed these mutants and co-expressed them in bacteria with TRM6 (B360 and B428, respectively). In addition, we created a mutant, called trm6-416/trm61-255 (B429), which has the three mutations in trm6-416 combined with the three mutations in trm61-255. All of these enzymes also lacked in vitro Mtase activity (Figure 4B).Figure 4.

Bottom Line: Yeast strains expressing trm6 and trm61 mutants exhibited growth phenotypes indicative of reduced m1A formation.In addition, recombinant mutant enzymes had reduced in vitro Mtase activity.We demonstrate that the mutations introduced do not prevent heteroligomer formation and do not disrupt binding of the cofactor S-adenosyl-L-methionine.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Marquette University, P.O. Box 1881, Milwaukee, WI 53201, USA.

ABSTRACT
In Saccharomyces cerevisiae, a two-subunit methyltransferase (Mtase) encoded by the essential genes TRM6 and TRM61 is responsible for the formation of 1-methyladenosine, a modified nucleoside found at position 58 in tRNA that is critical for the stability of tRNA(Met)i The crystal structure of the homotetrameric m1A58 tRNA Mtase from Mycobacterium tuberculosis, TrmI, has been solved and was used as a template to build a model of the yeast m1A58 tRNA Mtase heterotetramer. We altered amino acids in TRM6 and TRM61 that were predicted to be important for the stability of the heteroligomer based on this model. Yeast strains expressing trm6 and trm61 mutants exhibited growth phenotypes indicative of reduced m1A formation. In addition, recombinant mutant enzymes had reduced in vitro Mtase activity. We demonstrate that the mutations introduced do not prevent heteroligomer formation and do not disrupt binding of the cofactor S-adenosyl-L-methionine. Instead, amino acid substitutions in either Trm6p or Trm61p destroy the ability of the yeast m1A58 tRNA Mtase to bind tRNA(Met)i, indicating that each subunit contributes to tRNA binding and suggesting a structural alteration of the substrate-binding pocket occurs when these mutations are present.

Show MeSH
Related in: MedlinePlus