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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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Mutations in either Trm6p or Trm61p abolish tRNA binding. (A) Various concentrations of purified recombinant wild-type enzyme were incubated with a constant amount of 32P end-labeled  (1 nM) purified from a trm6Δ strain (Y146) (7). Bound  was trapped on a nitrocellulose filter and measured using liquid scintillation. The percent bound was determined by dividing the amount of radiolabeled tRNA bound by the total amount of radiolabeled tRNA in each reaction. (B) Purified recombinant wild-type and mutant enzymes were tested for tRNA binding as in (A), but with 500 nM protein. The percent  bound is reported as a percentage of that bound by the wild-type enzyme, which was set to 100%, corresponding to ∼30% of the input bound. The data reported is the average of duplicate trials.
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Figure 6: Mutations in either Trm6p or Trm61p abolish tRNA binding. (A) Various concentrations of purified recombinant wild-type enzyme were incubated with a constant amount of 32P end-labeled (1 nM) purified from a trm6Δ strain (Y146) (7). Bound was trapped on a nitrocellulose filter and measured using liquid scintillation. The percent bound was determined by dividing the amount of radiolabeled tRNA bound by the total amount of radiolabeled tRNA in each reaction. (B) Purified recombinant wild-type and mutant enzymes were tested for tRNA binding as in (A), but with 500 nM protein. The percent bound is reported as a percentage of that bound by the wild-type enzyme, which was set to 100%, corresponding to ∼30% of the input bound. The data reported is the average of duplicate trials.

Mentions: Because mutations made in Trm6p that are predicted to be located at the Trm6p/Trm61p interface did not affect oligomerization or AdoMet binding, we wanted to determine whether or not tRNA binding was altered. First, increasing amounts of purified recombinant wild-type enzyme were incubated with radiolabeled purified from a trm6Δ strain (Y146) (7). bound by Trm6p/Trm61p complexes was trapped on a nitrocellulose filter and quantitated by liquid scintillation counting. A binding curve generated from this data (Figure 6A) was used to determine the Kd for tRNA to be 330 ± 80 nM. To test the tRNA-binding ability of trm6 mutant enzymes, purified enzymes (500 nM) were assayed as described above. The trm6-416, trm6-420 and trm6-504 mutants did not bind considerably more tRNA than a control reaction lacking enzyme (Figure 6B). In addition, we did not detect tRNA binding when the concentration of trm6-416 and trm6-420 complexes used was increased 10-fold (5 μM), and, therefore, could not create binding curves for these enzymes. Because the mutations in trm61-255 and trm6-416/trm61-255 are predicted to lie in a structurally similar region as the trm6 mutations, and as these enzymes form oligomers but lack activity, we tested their ability to bind tRNA. Similar to trm6-416, trm6-420 and trm6-504 mutants, the trm61-255 and trm6-416/trm61-255 mutants did not bind (Figure 6B). Importantly, the trm61-3 mutant, which we have shown is defective in AdoMet binding, is able to bind as effectively as the wild-type enzyme. We conclude that the reduced m1A levels observed in vivo and the lack of Mtase activity seen in vitro result from the inability of trm6-416, trm6-420, trm6-504, trm61-255 and trm6-416/trm61-255 mutants to effectively bind their tRNA substrate.Figure 6.


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)

Mutations in either Trm6p or Trm61p abolish tRNA binding. (A) Various concentrations of purified recombinant wild-type enzyme were incubated with a constant amount of 32P end-labeled  (1 nM) purified from a trm6Δ strain (Y146) (7). Bound  was trapped on a nitrocellulose filter and measured using liquid scintillation. The percent bound was determined by dividing the amount of radiolabeled tRNA bound by the total amount of radiolabeled tRNA in each reaction. (B) Purified recombinant wild-type and mutant enzymes were tested for tRNA binding as in (A), but with 500 nM protein. The percent  bound is reported as a percentage of that bound by the wild-type enzyme, which was set to 100%, corresponding to ∼30% of the input bound. The data reported is the average of duplicate trials.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: Mutations in either Trm6p or Trm61p abolish tRNA binding. (A) Various concentrations of purified recombinant wild-type enzyme were incubated with a constant amount of 32P end-labeled (1 nM) purified from a trm6Δ strain (Y146) (7). Bound was trapped on a nitrocellulose filter and measured using liquid scintillation. The percent bound was determined by dividing the amount of radiolabeled tRNA bound by the total amount of radiolabeled tRNA in each reaction. (B) Purified recombinant wild-type and mutant enzymes were tested for tRNA binding as in (A), but with 500 nM protein. The percent bound is reported as a percentage of that bound by the wild-type enzyme, which was set to 100%, corresponding to ∼30% of the input bound. The data reported is the average of duplicate trials.
Mentions: Because mutations made in Trm6p that are predicted to be located at the Trm6p/Trm61p interface did not affect oligomerization or AdoMet binding, we wanted to determine whether or not tRNA binding was altered. First, increasing amounts of purified recombinant wild-type enzyme were incubated with radiolabeled purified from a trm6Δ strain (Y146) (7). bound by Trm6p/Trm61p complexes was trapped on a nitrocellulose filter and quantitated by liquid scintillation counting. A binding curve generated from this data (Figure 6A) was used to determine the Kd for tRNA to be 330 ± 80 nM. To test the tRNA-binding ability of trm6 mutant enzymes, purified enzymes (500 nM) were assayed as described above. The trm6-416, trm6-420 and trm6-504 mutants did not bind considerably more tRNA than a control reaction lacking enzyme (Figure 6B). In addition, we did not detect tRNA binding when the concentration of trm6-416 and trm6-420 complexes used was increased 10-fold (5 μM), and, therefore, could not create binding curves for these enzymes. Because the mutations in trm61-255 and trm6-416/trm61-255 are predicted to lie in a structurally similar region as the trm6 mutations, and as these enzymes form oligomers but lack activity, we tested their ability to bind tRNA. Similar to trm6-416, trm6-420 and trm6-504 mutants, the trm61-255 and trm6-416/trm61-255 mutants did not bind (Figure 6B). Importantly, the trm61-3 mutant, which we have shown is defective in AdoMet binding, is able to bind as effectively as the wild-type enzyme. We conclude that the reduced m1A levels observed in vivo and the lack of Mtase activity seen in vitro result from the inability of trm6-416, trm6-420, trm6-504, trm61-255 and trm6-416/trm61-255 mutants to effectively bind their tRNA substrate.Figure 6.

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