Recombinant Drosophila mojavensis Cytoplasmic tRNA 2-thiolation protein 2 (GI19780)

What is the primary function of Cytoplasmic tRNA 2-thiolation protein 2 in Drosophila mojavensis?

Cytoplasmic tRNA 2-thiolation protein 2 plays a central role in the 2-thiolation of mcm(5)S(2)U at tRNA wobble positions, particularly in tRNA(Lys), tRNA(Glu), and tRNA(Gln). The protein likely forms a heterodimer with NCS6/CTU1 that ligates sulfur from thiocarboxylated URM1 onto the uridine of tRNAs at wobble positions . This modification is crucial for proper decoding of mRNA by the ribosome, as it helps restrict wobble in split codon boxes and enables efficient codon-anticodon interactions .

What are the structural characteristics of this protein?

The Drosophila mojavensis Cytoplasmic tRNA 2-thiolation protein 2 is a full-length protein consisting of 404 amino acids with a sequence that includes conserved domains characteristic of the CTU2/NCS2 family . The protein has a molecular weight of approximately 44.3 kDa (based on related proteins in the same family) . Its sequence contains critical motifs necessary for interaction with its partner proteins and for facilitating the thiolation reaction.

How evolutionarily conserved is the tRNA 2-thiolation system across species?

The tRNA 2-thiolation system is highly conserved across eukaryotes. Research demonstrates functional homologs across diverse species including yeast (NCS2), plants (CTU2), nematodes (C. elegans), and various Drosophila species . The Ctu1-Ctu2 (cytosolic thiouridylase) complex responsible for 2-thiolation of cytosolic tRNAs is particularly well-conserved, indicating the fundamental importance of this modification in translation across evolutionary lines . In plants, the homologs ROL5 (of yeast NCS6) and CTU2 (of yeast NCS2) form a complex essential for tRNA thiolation .

What are the recommended storage and handling conditions for recombinant GI19780?

For optimal activity and stability, store recombinant Drosophila mojavensis Cytoplasmic tRNA 2-thiolation protein 2 at -20°C, and for extended storage, conserve at -20°C or -80°C. Avoid repeated freezing and thawing cycles; instead, prepare working aliquots and store at 4°C for up to one week . When reconstituting the protein, it is advisable to briefly centrifuge the vial before opening and reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Adding glycerol to a final concentration of 5-50% (with 50% being the standard recommendation) before aliquoting for long-term storage can help maintain stability .

How can I detect and quantify tRNA thiolation in experimental systems?

Two primary methods are used to detect tRNA thiolation:

• HPLC-MS (High-Performance Liquid Chromatography coupled with Mass Spectrometry): This technique can directly measure mcm(5)S(2)U and mcm(5)U levels in samples. In wild-type organisms, mcm(5)S(2)U is detected as the protonated molecule (MH+) and the protonated free base (BH2+) with expected m/z values of 333 and 201, while the unmodified mcm(5)U shows m/z values of 317 and 185 for MH+ and BH2+ respectively .

• APM/PAGE band shift method: This approach relies on the strong retardation of thiolated tRNA during electrophoresis due to the affinity of the thio group with the mercuric compound (APM) in the gel. This method can specifically identify thiolated tRNAs such as tRNA(Lys), which shows a characteristic band shift not seen in non-thiolated tRNAs like tRNA(Met) .

What protocols are recommended for protein-protein interaction studies involving GI19780?

To study protein-protein interactions of GI19780, consider these methodological approaches:

• Co-immunoprecipitation: Using antibodies against GI19780 or its potential interacting partners (particularly proteins in the CTU1/NCS6 family) to pull down protein complexes.

• Yeast two-hybrid assays: Based on the evidence that these proteins form functional complexes, Y2H is an effective screening tool to identify and confirm interactions with other proteins in the tRNA modification pathway.

• Bimolecular Fluorescence Complementation (BiFC): To visualize interactions in cellular contexts.

For the Drosophila mojavensis protein specifically, it's important to account for potential species-specific optimization of expression conditions, considering that the protein is typically expressed in yeast systems for recombinant production .

How does tRNA thiolation affect translation efficiency and fidelity?

tRNA thiolation at wobble positions plays a critical role in translation by:

• Codon Recognition Precision: The 2-thiolation of uridine at the first anticodon position (U34) is proposed to be crucial for restricting wobble in the corresponding split codon box, ensuring proper mRNA decoding .

• Translation Fidelity: Loss of thiolation can lead to both misreading and frame shifting during translation. Research in fission yeast shows that defects resulting from unmodified tRNAs can lead to severe genome instability .

• Selective Translation: In Arabidopsis, tRNA thiolation affects the translation of specific proteins including NPR1, a master immune regulator, demonstrating that this modification can selectively influence the translation of particular mRNAs rather than affecting global translation equally .

Experimental approaches to study these effects include ribosome profiling, polysome profiling, and reporter assays with codons dependent on thiolated tRNAs for efficient translation.

What are the implications of GI19780 dysfunction in cellular stress responses?

While direct evidence for GI19780 in stress responses is limited in the provided search results, research on related proteins in other organisms provides valuable insights:

• Thermosensitivity: Mutations in the Ctu1-Ctu2 complex lead to thermosensitive decrease of viability, suggesting a role in temperature stress adaptation .

• Genome Stability: Inactivation of the thiolation complex leads to marked ploidy abnormalities, indicating that proper tRNA modification is essential for maintaining genome integrity under stress conditions .

• Immune Response Regulation: In plants, the thiolation system is essential for immunity against pathogen infections, with mutations resulting in compromised immune responses .

These findings suggest that GI19780 dysfunction might similarly affect stress response pathways in Drosophila, particularly through altered translation of stress-responsive proteins.

How can CRISPR-Cas9 be applied to study GI19780 function in Drosophila models?

CRISPR-Cas9 technology offers powerful approaches to investigate GI19780 function:

• Gene Knockouts: Creating complete loss-of-function mutants similar to the rol5-c model described in plants, where a 2 bp deletion caused a frameshift .

• Domain-Specific Mutations: Introducing targeted mutations in functional domains to assess their specific contributions to protein activity.

• Tagged Variants: Creating endogenously tagged versions of GI19780 to track its localization and dynamics in vivo.

• Regulatory Element Modification: Altering promoter or enhancer regions to study expression regulation.

When designing sgRNAs, focus on conserved functional domains identified through comparison with homologs in other species. For phenotypic analysis, consider examining developmental timing, stress responses, and lifespan, as mutations in related proteins cause aberrant development and decreased viability .

What phenotypes are associated with GI19780 mutations in model organisms?

Based on studies of homologous proteins in various organisms, mutations in tRNA 2-thiolation proteins typically result in:

• Thermosensitive Viability: Organisms with mutations in the thiolation complex show decreased viability particularly at higher temperatures .

• Developmental Abnormalities: Aberrant development has been observed in multiple species with defects in this pathway .

• Genome Instability: Marked ploidy abnormalities occur in fission yeast with inactivated thiolation complexes .

• Immune Deficiency: In plants, mutation of the homologous ROL5 gene results in hyper-susceptibility to bacterial pathogens, demonstrating compromised immune function .

• Transcriptome and Proteome Dysregulation: Loss of thiolation affects both transcriptome and proteome reprogramming, particularly during stress responses .

How can Drosophila models of GI19780 dysfunction inform human disease research?

While the search results don't directly address human disease connections, the high conservation of the tRNA thiolation pathway suggests valuable translational potential:

• Neurological Disorders: Since precise translation is critical for neuronal function, Drosophila models could help investigate links between tRNA modification defects and neurological conditions.

• Cancer Biology: The observed genome instability and ploidy abnormalities in thiolation-deficient yeast suggest potential connections to cancer mechanisms that could be modeled in Drosophila.

• Immunological Disorders: The demonstrated role of tRNA thiolation in plant immunity suggests similar pathways might influence immune function across species.

Drosophila models offer advantages including rapid generation time, powerful genetic tools, and less ethical constraints compared to mammalian models, making them valuable for initial mechanistic studies before translation to human disease contexts.

What expression systems are optimal for recombinant production of GI19780?

Based on the available information, yeast expression systems appear to be the preferred choice for recombinant production of Drosophila mojavensis Cytoplasmic tRNA 2-thiolation protein 2 . When considering expression systems:

• Yeast Systems: Provide appropriate eukaryotic post-translational modifications and folding machinery for this conserved protein, which naturally functions in a eukaryotic context.

• E. coli Systems: May be considered for high-yield production but might require optimization for proper folding of this eukaryotic protein.

• Insect Cell Systems: Given the protein's origin in Drosophila, baculovirus-infected insect cells might provide the most native-like environment for expression.

For optimal results, include appropriate tags for purification while ensuring these don't interfere with protein function. The tag type should be determined during the manufacturing process based on specific experimental needs .

What are the critical factors for successful reconstitution and activity assays?

Several factors are critical for successful reconstitution and activity assessment:

• Buffer Composition: Use deionized sterile water for initial reconstitution to a concentration of 0.1-1.0 mg/mL . The buffer choice for downstream applications should maintain protein stability while supporting enzymatic activity.

• Storage Considerations: Add glycerol to a final concentration of 5-50% before aliquoting for long-term storage . For working aliquots, store at 4°C for up to one week to minimize freeze-thaw cycles.

• Activity Assays: To assess thiolation activity, consider:

  • In vitro thiolation of synthetic tRNA substrates

  • APM/PAGE band shift method to detect thiolated tRNAs

  • Mass spectrometry to identify thiolated nucleosides

• Partner Proteins: Since the protein likely functions as part of a complex with NCS6/CTU1 homologs , inclusion of appropriate partner proteins may be necessary for full enzymatic activity in reconstituted systems.

How can protein purity and integrity be verified following purification?

To ensure the quality of purified recombinant GI19780:

• SDS-PAGE Analysis: Verify protein size and purity, expecting a band at approximately 44.3 kDa. The protein should show >85% purity by SDS-PAGE as specified in product documentation .

• Western Blotting: Confirm protein identity using antibodies against the protein or included tags.

• Mass Spectrometry: For precise mass determination and to verify the absence of truncations or modifications.

• Activity Assays: Functional verification through in vitro thiolation assays or binding studies with known interaction partners.

• Circular Dichroism: To assess proper protein folding and secondary structure content.

When analyzing purity, be aware that the presence of tag sequences may alter the apparent molecular weight from the predicted 44.3 kDa. Particular attention should be paid to protein stability over time, as the shelf life in liquid form is typically 6 months at -20°C/-80°C, while the lyophilized form can remain stable for 12 months at the same temperatures .

How do the functional properties of GI19780 compare across Drosophila species?

The cytoplasmic tRNA 2-thiolation protein shows conservation across Drosophila species including D. mojavensis and D. ananassae , yet with species-specific sequence variations. While detailed comparative functional studies are not explicitly covered in the search results, analysis of the available sequences suggests:

• Core Functional Domains: Essential domains for thiolation activity and protein-protein interactions are likely highly conserved between species.

• Species-Specific Adaptations: Sequence variations may reflect adaptations to different environmental niches of various Drosophila species.

• Interaction Networks: Partner protein binding interfaces are expected to show co-evolution to maintain functional interactions across species.

A methodological approach to studying these differences would include comparative biochemical assays using recombinant proteins from different species, cross-species complementation experiments, and systematic mutagenesis of divergent residues to assess their functional significance.

What insights does phylogenetic analysis provide about tRNA thiolation across eukaryotes?

Phylogenetic analysis of tRNA thiolation machinery across eukaryotes reveals:

• Evolutionary Conservation: The CTU2/NCS2 family proteins, including GI19780, are highly conserved from yeast to plants and animals, indicating the fundamental importance of this modification system .

• Functional Adaptations: Despite this conservation, species-specific adaptations have occurred, as evidenced by the specialized roles in plant immunity discovered for the Arabidopsis homologs .

• Complex Formation: The interaction between CTU2-like proteins and their CTU1/NCS6-like partners appears to be an ancient and conserved feature, as similar complexes function in organisms from yeast to plants and insects .

This evolutionary conservation underscores the critical nature of tRNA thiolation for accurate translation across eukaryotic life and suggests that fundamental mechanisms discovered in one organism are likely applicable to others, including humans.

What new technologies might advance our understanding of tRNA thiolation mechanisms?

Several emerging technologies hold promise for advancing tRNA thiolation research:

• Cryo-EM for Complex Structures: High-resolution structural determination of the entire thiolation complex in action could reveal mechanistic details of the thiolation process.

• Single-molecule Fluorescence Techniques: To observe the dynamics of tRNA modification in real-time and potentially identify regulatory mechanisms.

• Nanopore Direct RNA Sequencing: For comprehensive detection of modified nucleosides in native tRNAs without amplification biases.

• Ribosome Profiling Combined with tRNA Modification Analysis: To directly correlate specific tRNA modifications with translation efficiency at individual codons.

• Computational Prediction Tools: Developing algorithms to predict the effects of tRNA modifications on translation efficiency and accuracy based on mRNA features.

These approaches could help address outstanding questions about how tRNA thiolation is regulated in response to cellular conditions and how it selectively affects the translation of specific mRNAs.

What are the unexplored aspects of GI19780 in cellular metabolism and stress adaptation?

Several key areas remain unexplored regarding GI19780's broader roles:

• Metabolic Sensing: Whether tRNA thiolation acts as a metabolic sensor, linking translation to cellular energetic status.

• Cell Cycle Regulation: Given the ploidy abnormalities observed in thiolation-deficient yeast , potential connections to cell cycle checkpoints deserve investigation.

• Cross-talk with Other tRNA Modifications: How thiolation interacts with other tRNA modifications to fine-tune translation.

• Tissue-Specific Functions: Potential differential roles in various tissues during Drosophila development and stress responses.

• Environmental Adaptation: Whether tRNA thiolation levels respond to environmental stimuli beyond temperature, such as nutrient availability or oxidative stress.