Recombinant Ashbya gossypii tRNA (uracil-O (2)-)-methyltransferase (TRM44), partial

What is TRM44 and what is its primary function in tRNA biology?

TRM44 is a tRNA (uracil-O(2)-)-methyltransferase that catalyzes the formation of 2'-O-methyluridine at position 44 (Um44) in specific tRNAs. In yeast, this enzyme is encoded by the YPL030w gene (renamed TRM44) and specifically modifies tRNA Ser species . The Um44 modification is highly conserved among eukaryotic cytoplasmic tRNAs with a long variable loop and is unique to tRNA Ser in yeast .

The modification occurs at a strategically important position in the tRNA structure - at the junction between the anticodon stem and the variable loop. Residue 44 forms tertiary interactions with m2,2G26 at the junction of the D-stem and anticodon stem, suggesting that Um44 plays a significant role in maintaining tRNA structural integrity .

Methodologically, the function of TRM44 was established through genetic deletion studies combined with tRNA isolation and HPLC analysis to detect specific modifications, clearly demonstrating that TRM44 is both necessary and sufficient for Um44 formation in tRNA Ser species .

How was TRM44 initially identified and characterized?

The identification of TRM44 involved a systematic functional genomics approach:

• Initial screening was performed using a yeast genomic library of affinity-purified GST-ORF fusion proteins

• Researchers used tRNA Ser(UGA) specifically labeled at the 3' phosphate of U44 as the substrate

• S-adenosylmethionine was used as the methyl donor

• Products were analyzed by digestion with RNase T1, RNase A, and phosphatase treatment

• Thin layer chromatography was used to resolve the resulting dinucleotide Um44pG from unreacted substrate

Verification of TRM44 function involved multiple complementary approaches:

• Showing that a trm44-Δ strain lacked 2'-O-methyltransferase activity

• Demonstrating that tRNA Ser isolated from trm44-Δ strains specifically lacked Um44 modification

• Proving that Trm44 purified from Escherichia coli could catalyze 2'-O-methylation of U44 in tRNA Ser in vitro

The reaction specifically required S-adenosylmethionine (SAM) as a methyl donor, with experiments showing complete absence of activity (<0.3%) when SAM was omitted .

What is the evolutionary conservation pattern of TRM44?

TRM44 exhibits an interesting evolutionary conservation pattern:

• Conserved among metazoans and fungi, consistent with the conservation of Um44 in eukaryotic tRNAs

• Surprisingly absent in plants, despite the functional importance of the modification

• Found in Ashbya gossypii (Eremothecium gossypii), a filamentous fungus phylogenetically related to S. cerevisiae

This evolutionary distribution pattern raises interesting questions about alternative mechanisms that might compensate for the absence of TRM44 in plants, or whether plants have evolved different tRNA stabilization strategies .

The high conservation of the Um44 modification itself is noteworthy:

• Present in 21 out of 23 characterized eukaryotic cytoplasmic tRNA Ser species

• Found in 10 of 22 characterized eukaryotic cytoplasmic tRNA Leu species

• Present in all five characterized mitochondrial tRNA Leu species from plants

• Found in mitochondrial tRNA Tyr from Tetrahymena pyriformis

This conservation pattern suggests an important functional role that has been maintained throughout eukaryotic evolution.

What experimental systems are available for studying recombinant Ashbya gossypii TRM44?

Several experimental systems have been developed for studying recombinant A. gossypii TRM44:

Expression Systems:

• Baculovirus expression system for producing recombinant protein with high purity (>85% as determined by SDS-PAGE)

• E. coli expression system for His6-tagged Trm44, which retains enzymatic activity

Available Research Tools:

• Recombinant TRM44 protein, available in both liquid and lyophilized forms

• Antibodies against TRM44, including rabbit polyclonal antibodies that have been shown to work in multiple applications (EIA, Immunoassay, ELISA, and Western Blot)

Storage and Handling Recommendations:

• Liquid form has a shelf life of approximately 6 months at -20°C/-80°C

• Lyophilized form has a shelf life of approximately 12 months at -20°C/-80°C

• Reconstitution should be performed in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Addition of 5-50% glycerol is recommended for long-term storage

• Repeated freezing and thawing should be avoided

For protein expression and purification, it's important to note that no dissociable cofactors are required for TRM44 activity under standard conditions, other than S-adenosylmethionine which is included in reaction mixtures .

What methodologies can be used to assay TRM44 methyltransferase activity?

The following methodological approaches have been developed to assay TRM44 activity:

Analysis of tRNA Modifications by HPLC:

To quantitatively analyze Um44 and other modifications in tRNA:

• Isolate specific tRNA species from cells

• Digest to nucleosides

• Resolve by HPLC

• Identify modifications based on retention times and UV absorbance spectra

• Quantify modification levels (mol/mol of tRNA)

Using this approach, researchers have shown that Um levels in tRNA Ser(IGA) are reduced from 0.84 mol/mole in wild-type strains to less than 0.01 mol in trm44-Δ strains .

How do genetic interactions between TRM44 and other tRNA modification genes affect cellular phenotypes?

Genetic interaction studies have revealed important insights about TRM44 function:

Key Genetic Interactions:

• Single trm44-Δ mutants show no observable growth defect across a range of temperatures (18°C to 37°C) in various media

• Double mutant trm44-Δ tan1-Δ strains (lacking both Um44 and ac4C12) exhibit temperature sensitivity at 33°C and above

• The synthetic growth defect in trm44-Δ tan1-Δ strains is complemented by introduction of either missing gene on a single-copy plasmid

Molecular Basis of the Phenotype:

The temperature sensitivity of trm44-Δ tan1-Δ double mutants arises from:

• Reduced levels of specific tRNA Ser species, particularly tRNA Ser(CGA) and tRNA Ser(UGA)

• The growth defect can be suppressed by introducing multiple copies of tRNA Ser(CGA) and tRNA Ser(UGA) genes

• The defect likely involves the rapid tRNA decay (RTD) pathway, as it can be suppressed by met22-Δ mutation

Implications for tRNA Quality Control:

This synthetic interaction demonstrates that:

• Multiple tRNA modifications cooperatively maintain tRNA stability

• Loss of specific combinations of modifications triggers quality control mechanisms

• The cell has surveillance systems (RTD pathway) that monitor tRNA structural integrity

These findings underscore the biological importance of tRNA modifications in maintaining functional tRNA pools and reveal how cells employ quality control mechanisms to eliminate structurally compromised tRNAs .

What role does TRM44 play in tRNA stability and quality control?

TRM44-mediated Um44 modification contributes significantly to tRNA quality control:

Rapid tRNA Decay (RTD) Pathway:

• The RTD pathway degrades mature tRNAs lacking certain modifications

• This pathway is mediated by 5'-3' exonucleases Rat1 and Xrn1

• Met22 regulates Rat1 and Xrn1 activity, likely through its substrate pAp

Evidence for TRM44's Role in tRNA Stability:

• tRNA Ser(CGA) and tRNA Ser(UGA) levels are specifically reduced in trm44-Δ tan1-Δ double mutants at elevated temperatures

• The temperature-sensitive phenotype of trm44-Δ tan1-Δ mutants is suppressed by met22-Δ mutation, which prevents RTD

• Enhanced RTD occurs in tef1-Δ derivative strains, leading to accelerated degradation of specific tRNAs

Mechanism of Destabilization:

The Um44 modification likely contributes to tRNA stability by:

• Maintaining proper tertiary structure at the junction between the anticodon stem and variable loop

• Facilitating interactions with m2,2G26 across from position 44

• Preventing recognition by quality control systems that target structurally compromised tRNAs

This role in quality control places TRM44 within a broader network of tRNA surveillance mechanisms that maintain the integrity of the cellular tRNA pool, removing damaged or improperly modified tRNAs that might otherwise impair translation fidelity .

What are the structural and biochemical characteristics of Ashbya gossypii TRM44?

The structural and biochemical properties of A. gossypii TRM44 include:

Protein Characteristics:

• Enzymatic classification: EC 2.1.1.211 (tRNA (uracil-O(2)-)-methyltransferase)

• Catalyzes the transfer of a methyl group from S-adenosylmethionine to the 2'-O position of uridine at position 44 in tRNA

• Requires S-adenosylmethionine (SAM) as a methyl donor

• When expressed in E. coli as a His6-fusion protein, it has a molecular weight of approximately 66 kDa

Substrate Specificity:

• In yeast, specifically modifies tRNA Ser species

• Recognizes tRNAs with a long variable loop

• Acts on position 44, which is at a crucial structural junction in the tRNA

Protein Production and Purification:

• Can be expressed as a recombinant protein in baculovirus or E. coli systems

• Purification can be achieved through affinity chromatography (e.g., IMAC for His-tagged versions)

• Retains activity after purification, suggesting no requirement for additional protein cofactors

The detailed structural data for A. gossypii TRM44 is still limited compared to other methyltransferases, presenting opportunities for further structural biology research to elucidate the precise mechanism of substrate recognition and catalysis.

How can Ashbya gossypii TRM44 be effectively expressed and purified for research applications?

Based on published methodologies, the following approaches are recommended for expression and purification of A. gossypii TRM44:

Expression Systems:

• Baculovirus Expression System:

  • Yields protein with >85% purity as determined by SDS-PAGE

  • Suitable for producing substantial quantities of functional protein

• E. coli Expression System:

  • Expression as a His6-tagged fusion protein

  • Demonstrated to yield enzymatically active protein

  • Allows for straightforward purification via immobilized metal affinity chromatography (IMAC)

Protein Handling:

• Store at -20°C or -80°C to maintain stability

• For long-term storage, add glycerol to a final concentration of 5-50%

• Avoid repeated freeze-thaw cycles

• When reconstituting lyophilized protein, use deionized sterile water to a concentration of 0.1-1.0 mg/mL

Activity Testing:

• Verify enzyme activity using the methyltransferase assay described earlier

• Check substrate specificity using different tRNA species

• Include appropriate controls (no enzyme, no SAM, heat-inactivated enzyme)

These methodologies provide a framework for researchers to obtain active recombinant A. gossypii TRM44 for various applications including enzymatic studies, structural analyses, and inhibitor screening.

What potential biotechnological applications involve Ashbya gossypii and its tRNA modification systems?

A. gossypii has emerging biotechnological applications that might benefit from understanding its tRNA modification systems:

Current Applications of A. gossypii:

• Industrial production of riboflavin

• Recently explored as a host system for recombinant protein production

• Platform for producing plant monoterpenes including sabinene, linalool, limonene, and pinene from agro-industrial wastes

• Efficient utilization of xylose-rich feedstocks and mixed formulations of corn-cob lignocellulosic hydrolysates with sugarcane or beet molasses

Potential Relevance of tRNA Modifications:

• tRNA modifications like those mediated by TRM44 can affect translation efficiency and fidelity

• Manipulating tRNA modification pathways could potentially optimize protein expression

• Understanding tRNA quality control mechanisms might help improve stress tolerance and productivity of industrial strains

Specific Achievements in A. gossypii Engineering:

Using engineered A. gossypii strains with modified metabolic pathways, researchers have achieved:

• Limonene production of 383 mg/L

• Sabinene production of 684.5 mg/L (representing a significant improvement compared to other organisms in flask culture mode)

These achievements illustrate A. gossypii's potential as a versatile industrial organism. While direct applications of TRM44 modification in biotechnology are not yet reported, the fundamental understanding of tRNA biology in this organism could contribute to future strain optimization strategies.

What methodological approaches can be used to study the impact of TRM44 deficiency on cellular tRNA pools?

Several sophisticated methodologies have been employed to study how TRM44 deficiency affects tRNA pools:

Quantitative Analysis of tRNA Species:

• HPLC Analysis of tRNA Modifications:

  • Purify specific tRNA species from wild-type and mutant strains

  • Digest to nucleosides and separate by HPLC

  • Identify modifications based on retention times and UV spectra

  • Quantify modification levels in mol/mol of tRNA

• Northern Blot Analysis for tRNA Levels:

  • Extract total RNA from cells under different conditions

  • Resolve by polyacrylamide gel electrophoresis

  • Transfer to membrane and probe with tRNA-specific oligonucleotides

  • Quantify relative levels of specific tRNA species

Genetic Approaches:

• Suppressor Analysis:

  • Introduce multiple copies of specific tRNA genes to test rescue of phenotypes

  • Create triple mutants (e.g., trm44-Δ tan1-Δ met22-Δ) to test pathway involvement

  • Perform genetic screens for suppressors of growth defects

• Temperature Shift Experiments:

  • Grow cells at permissive temperature then shift to non-permissive temperature

  • Monitor kinetics of tRNA decay after temperature shift

  • Compare rates between different mutant combinations

Functional Assessment:

• Charging State Analysis:

  • Separate charged from uncharged tRNAs using acidic gel electrophoresis

  • Monitor changes in charging levels after temperature shifts or stress conditions

  • Correlate with growth defects and translation efficiency

These approaches have collectively revealed that:

• trm44-Δ tan1-Δ mutants show reduced levels of tRNA Ser(CGA) and tRNA Ser(UGA) at elevated temperatures

• The RTD pathway is responsible for the accelerated degradation of these tRNAs

• This degradation can be enhanced in tef1-Δ backgrounds or suppressed by met22-Δ mutation

How does recombinant TRM44 compare between Ashbya gossypii and Saccharomyces cerevisiae?

Comparing TRM44 between these related fungal species reveals important similarities and differences:

Similarities:

• Both function as tRNA (uracil-O(2)-)-methyltransferases catalyzing the formation of Um44 in tRNA

• Both require S-adenosylmethionine as methyl donor

• Both are conserved among fungi, consistent with the conservation of Um44 in eukaryotic tRNAs

• Both can be expressed as recombinant proteins that retain enzymatic activity

Functional Conservation:

• A. gossypii, like S. cerevisiae, is a filamentous Saccharomycete

• The close phylogenetic relationship suggests similar tRNA modification patterns

• Both organisms likely employ similar tRNA quality control mechanisms

Research Tools Available:

• For S. cerevisiae TRM44: Extensive genetic analyses, in vitro activity assays, and phenotypic characterization

• For A. gossypii TRM44: Commercially available recombinant protein and antibodies for research applications