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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 Trm6p and Trm61p do not prevent oligomerization. (A). Purified recombinant enzymes were fractionated using Superose 12 gel filtration chromatography and visualized using SDS-PAGE followed by Coomassie blue staining. (B) Whole cell yeast extract from a strain expressing either TRM6 (Y353), trm6-416 (Y354), trm6-504 (Y360) or trm6-420 (Y367) was fractionated using Superose 12 gel filtration chromatography. Fractions were analyzed using SDS-PAGE followed by western blotting with antibodies to either Trm6p or Trm61p. Positions of molecular weight standards (Apoferritin, 443 kDa; β-Amylase, 200 kDa; Alcohol Dehydrogenase, 150 kDa; Phosphorylase b, 100 kDa) are indicated along the top of the figures.
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Figure 5: Mutations in Trm6p and Trm61p do not prevent oligomerization. (A). Purified recombinant enzymes were fractionated using Superose 12 gel filtration chromatography and visualized using SDS-PAGE followed by Coomassie blue staining. (B) Whole cell yeast extract from a strain expressing either TRM6 (Y353), trm6-416 (Y354), trm6-504 (Y360) or trm6-420 (Y367) was fractionated using Superose 12 gel filtration chromatography. Fractions were analyzed using SDS-PAGE followed by western blotting with antibodies to either Trm6p or Trm61p. Positions of molecular weight standards (Apoferritin, 443 kDa; β-Amylase, 200 kDa; Alcohol Dehydrogenase, 150 kDa; Phosphorylase b, 100 kDa) are indicated along the top of the figures.

Mentions: Based on its crystal structure, the m1A58 Mtase from M. tuberculosis was described as a dimer of two dimers (12). The interactions between the two subunits that form one dimer are extensive, while the interactions between all four subunits are limited to a central barrel structure (12). As described above, we created alanine substitutions in Trm6p and Trm61p in order to destabilize protein–protein interactions and found that the mutant enzymes had reduced Mtase activity, although all of the mutant enzymes were still capable of forming heterodimers (Figure 4A). We wanted to determine whether this loss of activity was due to disruption of the predicted heterotetrameric structure of the enzyme. To size mutant Trm6p/Trm61p complexes, purified recombinant enzyme was analyzed by gel filtration chromatography. The fractions that contained Trm6p and Trm61p were determined using SDS-PAGE followed by Coomassie blue staining. Since the molecular weight of Trm6p is 55 kDa, and that of Trm61p is 44 kDa, a heterotetramer would be ∼200 kDa. The elution pattern of Trm6p/Trm61p complexes was consistent with formation of a heteroligomer, possibly a heterotetramer. Furthermore, no differences were seen between the elution profiles of the wild-type and trm6-416, trm6-420 and trm6-504 enzymes (data not shown), which led us to hypothesize that the substitutions in the Trm6p mutants were not enough to destabilize the predicted tetramer because other interactions provided by amino acids on Trm61p remained intact. Therefore, we also analyzed the trm61-255 mutant and the trm6-416/trm61-255 double mutant. We purified recombinant wild-type and Trm6-416p/Trm61p, Trm6p/Trm61-255p and Trm6-416p/Trm61-255p mutant complexes and used gel filtration chromatography to determine the sizes of these enzymes. All three mutant enzymes fractionated the same as the wild-type enzyme (Figure 5A). In conclusion, none of the mutations made in Trm6p and Trm61p to the predicted protein–protein interface of the yeast Mtase disrupted oligomerization.Figure 5.


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 Trm6p and Trm61p do not prevent oligomerization. (A). Purified recombinant enzymes were fractionated using Superose 12 gel filtration chromatography and visualized using SDS-PAGE followed by Coomassie blue staining. (B) Whole cell yeast extract from a strain expressing either TRM6 (Y353), trm6-416 (Y354), trm6-504 (Y360) or trm6-420 (Y367) was fractionated using Superose 12 gel filtration chromatography. Fractions were analyzed using SDS-PAGE followed by western blotting with antibodies to either Trm6p or Trm61p. Positions of molecular weight standards (Apoferritin, 443 kDa; β-Amylase, 200 kDa; Alcohol Dehydrogenase, 150 kDa; Phosphorylase b, 100 kDa) are indicated along the top of the figures.
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Related In: Results  -  Collection

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Figure 5: Mutations in Trm6p and Trm61p do not prevent oligomerization. (A). Purified recombinant enzymes were fractionated using Superose 12 gel filtration chromatography and visualized using SDS-PAGE followed by Coomassie blue staining. (B) Whole cell yeast extract from a strain expressing either TRM6 (Y353), trm6-416 (Y354), trm6-504 (Y360) or trm6-420 (Y367) was fractionated using Superose 12 gel filtration chromatography. Fractions were analyzed using SDS-PAGE followed by western blotting with antibodies to either Trm6p or Trm61p. Positions of molecular weight standards (Apoferritin, 443 kDa; β-Amylase, 200 kDa; Alcohol Dehydrogenase, 150 kDa; Phosphorylase b, 100 kDa) are indicated along the top of the figures.
Mentions: Based on its crystal structure, the m1A58 Mtase from M. tuberculosis was described as a dimer of two dimers (12). The interactions between the two subunits that form one dimer are extensive, while the interactions between all four subunits are limited to a central barrel structure (12). As described above, we created alanine substitutions in Trm6p and Trm61p in order to destabilize protein–protein interactions and found that the mutant enzymes had reduced Mtase activity, although all of the mutant enzymes were still capable of forming heterodimers (Figure 4A). We wanted to determine whether this loss of activity was due to disruption of the predicted heterotetrameric structure of the enzyme. To size mutant Trm6p/Trm61p complexes, purified recombinant enzyme was analyzed by gel filtration chromatography. The fractions that contained Trm6p and Trm61p were determined using SDS-PAGE followed by Coomassie blue staining. Since the molecular weight of Trm6p is 55 kDa, and that of Trm61p is 44 kDa, a heterotetramer would be ∼200 kDa. The elution pattern of Trm6p/Trm61p complexes was consistent with formation of a heteroligomer, possibly a heterotetramer. Furthermore, no differences were seen between the elution profiles of the wild-type and trm6-416, trm6-420 and trm6-504 enzymes (data not shown), which led us to hypothesize that the substitutions in the Trm6p mutants were not enough to destabilize the predicted tetramer because other interactions provided by amino acids on Trm61p remained intact. Therefore, we also analyzed the trm61-255 mutant and the trm6-416/trm61-255 double mutant. We purified recombinant wild-type and Trm6-416p/Trm61p, Trm6p/Trm61-255p and Trm6-416p/Trm61-255p mutant complexes and used gel filtration chromatography to determine the sizes of these enzymes. All three mutant enzymes fractionated the same as the wild-type enzyme (Figure 5A). In conclusion, none of the mutations made in Trm6p and Trm61p to the predicted protein–protein interface of the yeast Mtase disrupted oligomerization.Figure 5.

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