Recombinant Drosophila pseudoobscura pseudoobscura Eukaryotic translation initiation factor 3 subunit B (eIF3-S9), partial

What is the function of eIF3-S9 in Drosophila pseudoobscura pseudoobscura?

eIF3-S9 (eukaryotic translation initiation factor 3 subunit B) in D. pseudoobscura pseudoobscura functions as a crucial component of the translation initiation complex. As part of the larger eIF3 complex, it mediates the assembly of the 43S pre-initiation complex by facilitating the interaction between mRNA and the 40S ribosomal subunit. This protein is characterized by alternative identifiers including DpseGA24735, dpse_GLEANR_15408, and GA24735 in genomic databases .

To investigate this function experimentally, researchers should consider:

• In vitro translation assays using purified components with and without eIF3-S9

• RNAi-mediated knockdown studies in D. pseudoobscura cell lines

• Co-immunoprecipitation experiments to identify interaction partners

• Polysome profiling to assess effects on global translation

The importance of eIF3-S9 extends beyond basic translation, as it may play specialized roles in development and stress response pathways specific to Drosophila species.

How does eIF3-S9 expression pattern correlate with developmental stages in D. pseudoobscura?

The expression pattern of eIF3-S9 in D. pseudoobscura shows distinct developmental regulation that recapitulates phylogenetic relationships among Drosophila species. Gene expression analysis has identified eIF3-S9 as one of the genes whose temporal expression profiles correlate with the known Drosophila phylogeny, suggesting evolutionary constraints on its expression .

Appropriate methods for studying developmental expression include:

• Quantitative RT-PCR with tight developmental time-course sampling

• RNA-seq analysis across embryonic, larval, pupal, and adult stages

• Western blotting with stage-specific antibodies

• In situ hybridization to determine tissue-specific expression patterns

When analyzing expression data, researchers should normalize against established housekeeping genes and consider examining multiple D. pseudoobscura strains to account for natural variation.

What are the optimal conditions for storing and handling recombinant D. pseudoobscura eIF3-S9?

For maintaining maximum activity of recombinant D. pseudoobscura eIF3-S9, researchers should adhere to these evidence-based storage and handling protocols:

Additional handling considerations:

• Avoid repeated freeze-thaw cycles as they significantly reduce activity

• The recombinant protein has ≥85% purity as determined by SDS-PAGE

• Use low-binding microcentrifuge tubes to prevent protein loss

• Include protease inhibitors when working with cell extracts

• Perform activity tests after long-term storage before experimental use

These storage conditions should be validated for each specific preparation, as purification methods and protein concentration may affect stability.

What expression systems are suitable for producing recombinant D. pseudoobscura eIF3-S9?

Multiple expression systems have been successfully employed for recombinant D. pseudoobscura eIF3-S9 production, each with specific advantages depending on experimental requirements:

According to product specifications, all four expression systems have been successfully used to produce this protein with ≥85% purity as determined by SDS-PAGE . The choice of expression system should be guided by the intended application, with E. coli being sufficient for basic binding studies and structural analysis, while insect or mammalian systems are preferred for functional assays where post-translational modifications are critical.

What are essential controls for experiments using recombinant D. pseudoobscura eIF3-S9?

When designing experiments with recombinant D. pseudoobscura eIF3-S9, implementing appropriate controls is crucial for result interpretation and validation:

Additional methodological considerations include:

• Verify protein identity by Western blotting or mass spectrometry

• Confirm proper folding using circular dichroism spectroscopy

• For cell-based assays, include mock-transfected cells

• When studying protein-protein interactions, include controls for non-specific binding

These controls ensure experimental rigor and reproducibility when working with recombinant D. pseudoobscura eIF3-S9.

How can structural analysis of D. pseudoobscura eIF3-S9 inform evolutionary studies?

Structural characterization of D. pseudoobscura eIF3-S9 provides valuable insights into evolutionary conservation and divergence of translation initiation mechanisms. A comprehensive structural analysis approach should include:

• Sequence conservation analysis across Drosophila species:

  • Multiple sequence alignment to identify conserved motifs

  • Calculation of selection pressure (dN/dS ratios) for different domains

  • Identification of lineage-specific insertions/deletions

• Homology modeling and structural prediction:

  • Template-based modeling using solved structures of eIF3-S9 homologs

  • Conservation mapping onto structural models

  • Identification of surface-exposed vs. buried residues

• Functional domain analysis:

  • RNA-binding motifs conservation

  • Protein interaction interfaces identification

  • Post-translational modification sites comparison

The correlation between gene expression patterns in Drosophila embryogenesis and phylogenetic relationships suggests evolutionary constraints on translation factors like eIF3-S9. Structural analysis can reveal whether these constraints manifest as structural conservation in specific functional domains or allow for structural plasticity in non-critical regions.

What approaches can be used to study protein-protein interactions of eIF3-S9 in the translation initiation complex?

Investigating the interactome of D. pseudoobscura eIF3-S9 requires multiple complementary approaches to capture both stable and transient interactions:

Implementation strategy:

• Start with affinity purification-mass spectrometry to identify the core interactome

• Validate key interactions using direct binding assays with purified recombinant proteins

• Map interaction interfaces through mutational analysis and crosslinking

• Visualize complexes using structural biology approaches

Expected interactors include other eIF3 subunits (particularly eIF3-S10, eIF3-S8, and eIF3-S5) , components of the 40S ribosomal subunit, and potentially mRNA-binding proteins specific to D. pseudoobscura.

How does the mutation rate of D. pseudoobscura affect research on eIF3-S9 evolution?

The mutation rate in D. pseudoobscura has significant implications for studies on eIF3-S9 evolution and function. Research has shown that spontaneous mutation rates in D. pseudoobscura vary significantly depending on genetic background and hybridization status:

This variation in mutation rates means researchers studying eIF3-S9 evolution should:

• Account for strain-specific differences when analyzing sequence variation

• Consider the potential for accelerated evolution in hybrid zones

• Compare synonymous vs. non-synonymous substitution rates to detect selection

• Examine intron vs. exon variation to distinguish neutral from functional changes

The significantly higher mutation rate in hybrids (p-value = 0.003) suggests that genes like eIF3-S9 might evolve faster in hybrid populations, potentially leading to functional innovations in translation regulation. When studying natural populations, researchers should document the precise origin of samples and consider potential hybridization history.

What techniques are appropriate for studying post-translational modifications of D. pseudoobscura eIF3-S9?

Post-translational modifications (PTMs) of eIF3-S9 play crucial roles in regulating its function. A comprehensive PTM analysis workflow should include:

Sample preparation considerations:

• Rapid extraction in the presence of phosphatase/protease inhibitors

• Enrichment steps for low-abundance modifications

• Comparison across developmental stages and stress conditions

• Use of recombinant protein with ≥85% purity for in vitro modification assays

This approach allows researchers to identify which PTMs regulate eIF3-S9 function during development or under stress conditions, and how these regulatory mechanisms might differ between D. pseudoobscura and other Drosophila species.

How can recombinant D. pseudoobscura eIF3-S9 be used to study stress response mechanisms?

Recombinant D. pseudoobscura eIF3-S9 serves as a valuable tool for investigating translation regulation during stress responses:

Research methodology:

• Pre-incubate recombinant eIF3-S9 with stress-activated kinases/modifying enzymes

• Analyze modification patterns using mass spectrometry or phospho-specific detection

• Test altered binding to other initiation factors and mRNA

• Compare with endogenous eIF3-S9 behavior in stressed cells

This approach reveals how post-translational modifications of eIF3-S9 under stress conditions alter its function in translation initiation, potentially promoting the selective translation of stress-response mRNAs.

The high mutation rate observed in D. pseudoobscura crosses suggests potential adaptation to different stressors, making it interesting to compare stress-responsive modifications of eIF3-S9 across different D. pseudoobscura populations and related species.

What are the key considerations for designing cross-species complementation assays with eIF3-S9?

Cross-species complementation assays provide powerful insights into functional conservation and divergence of eIF3-S9 across Drosophila species:

Experimental design framework:

• Generate eIF3-S9 knockdown or knockout in D. pseudoobscura cells using RNAi or CRISPR-Cas9

• Rescue with eIF3-S9 orthologs from multiple Drosophila species (D. melanogaster, D. persimilis, etc.)

• Measure rescue efficiency through multiple parameters:

• Create chimeric proteins with domains from different species to map functionally divergent regions

Critical controls:

• Empty vector negative control

• Wild-type D. pseudoobscura eIF3-S9 positive control

• Expression level normalization across constructs

• Testing under both normal and stress conditions

This approach can reveal the degree of functional conservation across evolutionary distances and identify species-specific adaptations in translation regulation mechanisms. Given that gene expression patterns in D. pseudoobscura development recapitulate phylogenetic relationships , this approach can determine whether functional conservation mirrors expression pattern conservation.

How can researchers optimize purification protocols for functionally active D. pseudoobscura eIF3-S9?

Obtaining pure, functionally active D. pseudoobscura eIF3-S9 requires optimized purification strategies:

Optimized purification protocol:

• Express with N-terminal His-tag and C-terminal StrepII-tag for tandem purification

• Lyse cells in buffer containing 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 5% glycerol, 1 mM DTT

• Perform initial IMAC purification with imidazole gradient elution

• Apply to StrepTactin column for second purification step

• Include final size-exclusion chromatography step

• Verify ≥85% purity by SDS-PAGE

• Confirm identity by mass spectrometry or Western blotting

• Test functionality through binding assays with other eIF3 subunits

This approach yields highly pure, functionally active protein suitable for biochemical and structural studies. The dual-tag strategy allows for removal of truncated products and contaminants that co-purify with single affinity steps.

What methodologies can elucidate the role of eIF3-S9 in mRNA-specific translation regulation?

Investigating the role of D. pseudoobscura eIF3-S9 in mRNA-specific translation regulation requires specialized approaches:

• RNA immunoprecipitation followed by sequencing (RIP-seq):

  • Immunoprecipitate eIF3-S9 from D. pseudoobscura cells

  • Isolate and sequence associated mRNAs

  • Identify enriched sequence or structural motifs

  • Compare with other Drosophila species to identify conserved targets

• Ribosome profiling with eIF3-S9 perturbation:

  • Deplete eIF3-S9 using RNAi or CRISPR

  • Perform ribosome footprinting

  • Analyze changes in translation efficiency genome-wide

  • Identify mRNAs differentially affected by eIF3-S9 loss

• In vitro translation assays:

  • Use purified recombinant eIF3-S9 (≥85% purity)

  • Test translation of reporter mRNAs with different 5' UTRs

  • Measure dependence on eIF3-S9 concentration

  • Identify structural features conferring eIF3-S9 dependence

• CLIP-seq (Crosslinking immunoprecipitation with sequencing):

  • Map direct RNA binding sites of eIF3-S9

  • Identify consensus binding motifs

  • Correlate binding with translation efficiency

  • Compare across stress conditions

These approaches reveal how eIF3-S9 contributes to selective mRNA translation, which is particularly relevant during developmental transitions and stress responses where gene expression profiles recapitulate phylogenetic relationships .