Recombinant Thermoplasma acidophilum tRNA (guanine (26)-N (2))-dimethyltransferase
Description
Enzymatic Function and Catalytic Mechanism
tRNA (guanine26-N2)-dimethyltransferase (Trm1) sequentially transfers two methyl groups from S-adenosyl-L-methionine (SAM) to the N2 position of guanine26 in tRNA, forming N2-methylguanosine (m2G26) and then N2,N2-dimethylguanosine (m²2G26) . The enzyme dissociates from the tRNA substrate after the first methylation, requiring rebinding to complete the second transfer . Key identity elements for dimethylation include:
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Base pairs C11●G24 and G10●C25 in the D-stem
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A five-nucleotide variable loop
Biochemical Properties
Research Findings and Comparative Analysis
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Modification Context: In T. acidophilum, m²2G26 coexists with unique tRNA modifications like N2,N2,2′-O-trimethylguanosine (m²2Gm26) and m7G49, mediated by distinct methyltransferases .
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Comparative Insights:
Role in tRNA Stability and Thermostability
m²2G26 stabilizes the tRNA’s D-arm and variable loop, preventing thermal denaturation. In T. acidophilum, this modification is critical for:
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Maintaining codon-anticodon fidelity at high temperatures.
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Facilitating proper tRNA processing and ribosome interactions .
Modified Nucleosides in Thermoplasma acidophilum tRNA
| Modified Nucleoside | Position | Enzyme Responsible | Functional Role |
|---|---|---|---|
| m²2G26 | 26 | Trm1 | Thermal stability |
| s⁴U8/s⁴U9 | 8/9 | ThiI | Redox protection |
| m7G49 | 49 | Unknown MT | Structural reinforcement |
| Cm56 | 56 | Trm56 | Ribose methylation |
Open Questions and Research Gaps
Product Specs
Note: We will prioritize shipping the format we have in stock. However, if you require a specific format, please indicate your preference when placing the order, and we will fulfill your request.
Note: All of our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance, as additional fees will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Target Background
KEGG: tac:Ta0997
STRING: 273075.Ta0997
Q&A
How is recombinant Thermoplasma acidophilum Trm1 expressed and purified for functional studies?
Recombinant Trm1 is typically produced in Escherichia coli with an N-terminal His6-tag for affinity purification. Key steps include:
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Cloning: Amplify the Trm1 gene (e.g., Ta0997 in T. acidophilum) and insert into a plasmid under a T7 promoter .
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Expression: Induce protein expression at low temperatures (e.g., 16°C) to enhance solubility .
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Purification: Use Ni-NTA chromatography followed by ion-exchange chromatography (e.g., HiTrap Q HP) and gel filtration (e.g., Superdex 200) to achieve homogeneity .
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Validation: Confirm activity using in vitro methylation assays with S-adenosyl-L-methionine (AdoMet) and tRNA transcripts .
What experimental conditions optimize Trm1-mediated tRNA methylation?
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Temperature: Optimal activity occurs at 50–95°C, reflecting the enzyme’s thermostability (half-life of 2 hours at 95°C) .
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Substrate ratio: A 1:1 enzyme-to-tRNA ratio ensures efficient dimethylation (m₂²G26 formation) .
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tRNA structure: Mature tRNA transcripts lacking introns are preferred, as intron-containing precursors show reduced methylation rates .
How is Trm1 activity quantified in vitro?
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Radiolabeling: Use ¹⁴C-AdoMet to track methyl group transfer .
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RNase T2 digestion: Digest methylated tRNA and analyze products via 2D thin-layer chromatography (2D-TLC) to distinguish m₂G26 and m₂²G26 .
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Mass spectrometry: Confirm modified nucleosides (e.g., m₂²G26 in T. acidophilum tRNAMet) .
What structural elements in tRNA dictate Trm1 substrate specificity?
Trm1 requires:
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Variable loop: A 5-nucleotide loop for proper 3D tRNA folding .
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Core stability: Disruption of tertiary interactions (e.g., loss of Ψ55 or m⁷G46) reduces methylation efficiency .
Table 1: Impact of tRNA structural mutations on Trm1 activity
| Mutation | m₂²G26 Yield | Proposed Mechanism |
|---|---|---|
| C11A | <10% | Loss of D-stem stability |
| Variable loop Δ2 | 15% | Impaired 3D core formation |
| Ψ55 deletion | 40% | Altered tRNA flexibility |
How does Trm1 dissociate between monomethylation (m₂G26) and dimethylation (m₂²G26)?
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Kinetic evidence: Trm1 releases tRNA after monomethylation, requiring rebinding for the second methyl transfer .
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Enzyme-tRNA ratio: Excess Trm1 promotes complete dimethylation, while limiting enzyme favors m₂G26 accumulation .
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Temperature dependence: Higher temperatures (e.g., 95°C) accelerate dissociation-reassociation cycles .
What regulatory networks link Trm1 to other tRNA modification enzymes?
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Cross-dependence: Trm1 activity is influenced by prior modifications (e.g., m⁷G46 by TrmB or Ψ55 by TruB) .
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Thermal adaptation: In Thermococcus kodakarensis, Trm1-mediated m₂²G26 stabilizes tRNA at 95°C, with Δtrm11 strains showing growth defects .
Table 2: Functional interplay between Trm1 and other enzymes
| Enzyme | Modification | Impact on Trm1 Activity |
|---|---|---|
| TrmB | m⁷G46 | Enhances tRNA stability |
| TruB | Ψ55 | Facilitates core folding |
| Trm14 | m²G6/m²G67 | Indirectly affects Trm1 |
How are contradictions in methylation site assignments resolved?
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Case study: Early reports identified G26 in T. acidophilum tRNAMet as m₂G, but LC-MS later revealed m₂²G26 .
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Resolution: Use nuclease digestion (e.g., RNase T2) combined with 2D-TLC or LC-MS/MS to differentiate mono- and dimethylated products .
Why do recombinant Trm1 enzymes show reduced activity compared to native forms?
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C-terminal truncations: The C-terminal domain in human Trm1 (absent in bacterial homologs) enhances RNA binding; deletions reduce in vivo activity .
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Solution: Optimize expression conditions (e.g., low-temperature induction) and include chaperones to improve folding .
Key Research Findings
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Evolutionary conservation: Trm1 homologs in Pyrococcus furiosus, T. acidophilum, and humans share a conserved catalytic core but differ in auxiliary domains .
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Biological role: m₂²G26 stabilizes tRNA tertiary structure, critical for thermophiles like T. acidophilum .
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Technical advance: Intron-containing pre-tRNAs can be methylated, but mature tRNAs are preferred substrates .
