Recombinant Ashbya gossypii ATP-dependent RNA helicase DBP10 (DBP10), partial

What is Ashbya gossypii ATP-dependent RNA helicase DBP10?

DBP10 is a DEAD-box RNA helicase (EC 3.6.4.13) found in the filamentous fungus Ashbya gossypii. It belongs to the larger family of DEAD-box proteins characterized by nine conserved sequence motifs, including the signature Walker B motif with the amino acid sequence D-E-A-D. Like other members of this protein family, DBP10 exhibits RNA-dependent ATPase activity and ATP-dependent RNA helicase functionality, playing a crucial role in restructuring pre-ribosomal RNA during ribosome biogenesis .

What biological role does DBP10 play in A. gossypii?

Based on functional studies of DBP10 homologs, this helicase appears to be critical for ribosome biogenesis in A. gossypii. Specifically, it likely functions in restructuring pre-ribosomal RNA of the evolving peptidyl-transferase center (PTC) on nucleolar ribosomal 60S assembly intermediates. Similar to its function in related organisms, DBP10 in A. gossypii likely facilitates proper folding of rRNA, association and dissociation of snoRNAs, and recruitment of ribosomal proteins during 60S ribosomal subunit assembly .

What expression systems are optimal for producing recombinant A. gossypii DBP10?

Multiple expression systems can be used for producing recombinant A. gossypii DBP10, each with distinct advantages:

The choice depends on experimental requirements, with baculovirus systems being particularly useful when maintaining protein functionality is critical .

What purification strategies yield the highest activity for recombinant DBP10?

For optimal purification of functionally active recombinant DBP10, a multi-step approach is recommended:

• Initial capture using affinity chromatography (typically His-tag or GST-tag based)

• Intermediate purification through ion exchange chromatography

• Polishing via size exclusion chromatography

Critical factors affecting purification success include:

• Maintaining RNA-free conditions during purification to prevent premature activation

• Including ATP or non-hydrolyzable ATP analogs in buffers to stabilize protein conformation

• Using optimized salt concentrations (typically 150-300 mM) to maintain solubility while reducing non-specific interactions

• Maintaining reducing conditions (1-5 mM DTT or 2-10 mM β-mercaptoethanol) to prevent oxidation of cysteine residues

• Performing purification at 4°C to minimize proteolytic degradation

Purification to >85% homogeneity (as assessed by SDS-PAGE) is generally sufficient for most research applications .

What biochemical assays can effectively measure DBP10 helicase activity?

Multiple complementary assays can be employed to characterize DBP10 helicase activity:

• RNA-dependent ATPase assay: Measures phosphate release using malachite green or radioactive ATP. This assay quantifies the RNA-stimulated ATP hydrolysis rate but does not directly measure RNA unwinding.

• RNA unwinding assay: Utilizes fluorescent or radiolabeled RNA duplexes to directly measure helicase activity. The substrates typically consist of a short RNA strand annealed to a longer one, with detection of unwinding through native gel electrophoresis.

• FRET-based unwinding assay: Employs RNA duplexes labeled with fluorophore pairs (donor and acceptor) to monitor unwinding in real-time.

• Surface plasmon resonance: Measures binding kinetics of DBP10 to RNA substrates and cofactors.

For DBP10 specifically, using ribosomal RNA structures or segments from the peptidyl-transferase center region as substrates would provide the most physiologically relevant activity measurements .

How can researchers differentiate between the RNA remodeling vs. protein displacement activities of DBP10?

Distinguishing between RNA remodeling and protein displacement requires specialized experimental approaches:

• For RNA remodeling activity:

  • Structure-specific RNA substrates with FRET pairs positioned to detect conformational changes rather than complete strand separation

  • Chemical probing techniques (SHAPE, DMS) to monitor RNA structural changes in the presence and absence of DBP10 and ATP

  • Native gel shift assays using structured RNA elements from the pre-rRNA

• For protein displacement activity:

  • RNP reconstitution assays with fluorescently labeled proteins

  • Co-immunoprecipitation experiments comparing protein association with pre-ribosomes in wild-type vs. catalytically inactive DBP10 mutants

  • Single-molecule approaches tracking protein dissociation in the presence of active DBP10

Recent studies on DEAD-box proteins suggest that seemingly small sequence variations can significantly impact substrate specificity and functional outcomes, making these detailed mechanistic studies particularly valuable .

Which conserved motifs should be targeted for structure-function studies of DBP10?

Strategic mutagenesis of specific conserved motifs can provide insights into different aspects of DBP10 function:

Evidence from studies of other DEAD-box helicases indicates that mutations within conserved catalytic helicase-core motifs can yield dominant-negative phenotypes, where the mutant protein stably associates with its substrate but fails to complete its function, thus blocking the process. For DBP10 specifically, such mutations have been shown to impair pre-60S biogenesis at the nucleolar stage .

How can researchers generate catalytically inactive DBP10 variants that maintain substrate binding?

To create catalytically inactive DBP10 variants that retain substrate binding capability:

• ATP binding but not hydrolysis:

  • E→Q substitution in the DEAD motif (Motif II)

  • This creates a "substrate trap" that binds but cannot release RNA

• Reduced ATP binding:

  • K→A substitution in the Walker A motif (AxTGxGKT)

  • This prevents ATP binding while maintaining protein structure

• Uncoupling ATP hydrolysis from helicase activity:

  • S→A substitution in Motif III (SAT)

  • This maintains ATP hydrolysis but prevents its coupling to unwinding

When designing these mutations, researchers should consider:

• Using site-directed mutagenesis with high-fidelity polymerases

• Confirming mutations by sequencing

• Verifying protein folding/stability through circular dichroism or thermal shift assays

• Testing substrate binding through electrophoretic mobility shift assays or surface plasmon resonance

These variants serve as valuable tools for mechanistic studies and for identifying interaction partners through pull-down experiments .

How does DBP10 coordinate with other factors during ribosome biogenesis?

DBP10 functions within a complex network of protein-protein and protein-RNA interactions during ribosome biogenesis:

• Temporal sequence of activities: DBP10 acts at a specific nucleolar stage of pre-60S assembly, with evidence indicating it functions prior to the release of assembly factor Rrp14 and stable integration of late nucleolar factors such as Noc3.

• Interaction with GTPases: DBP10 specifically interacts with the GTPase Nug1 through its N-terminal domain. This interaction appears critical, as mutations in DBP10 can inhibit Nug1 binding to pre-60S particles.

• Coordination with methyltransferases: DBP10 also coordinates with the methyltransferase Spb1, which methylates the 25S rRNA nucleotide G2922. When DBP10 function is impaired, Spb1 incorporation is reduced, resulting in decreased rRNA methylation.

• Restructuring of the peptidyl-transferase center: DBP10's helicase activity likely generates the necessary structural framework for assembly factor docking, thereby permitting proper rRNA modification and progression of pre-60S maturation.

This coordinated activity suggests that DBP10 functions as both an active restructuring enzyme and a scaffold that enables the sequential association of other ribosome assembly factors .

What phenotypes result from DBP10 mutations or depletion in A. gossypii?

While specific A. gossypii DBP10 depletion phenotypes are not directly described in the search results, extrapolating from studies in related organisms suggests:

• Ribosome biogenesis defects: Mutations in DBP10 likely cause accumulation of pre-60S ribosomal particles that fail to mature properly, resulting in nucleolar abnormalities and reduced mature ribosome production.

• Growth inhibition: As ribosome biogenesis is essential for cell growth, DBP10 mutations would likely cause significant growth defects, particularly under conditions demanding high protein synthesis rates.

• Dominant-negative effects: Catalytically inactive DBP10 mutants can exhibit dominant-lethal growth phenotypes by stably associating with pre-60S intermediates and blocking subsequent maturation steps.

• Specific rRNA processing defects: Defects in DBP10 function would likely result in accumulation of specific pre-rRNA intermediates that could be detected through northern blot analysis.

• Methylation deficiency: Reduced G2922 methylation in 25S rRNA would be expected, as DBP10 facilitates recruitment of the methyltransferase Spb1.

Complementation studies using homologs from other organisms could help determine the degree of functional conservation across species .

How conserved is DBP10 function across different fungal species?

DBP10 exhibits significant conservation across fungal species, reflecting its essential role in ribosome biogenesis:

• Sequence conservation: The core DEAD-box motifs are highly conserved, with variations primarily in the N and C-terminal regions that likely confer species-specific interactions.

• Functional conservation: The essential role in ribosome biogenesis appears to be maintained across species, from yeasts to filamentous fungi like A. gossypii.

• Evolutionary significance: The conservation of DBP10 across the fungal kingdom suggests strong selective pressure to maintain its function in ribosome assembly.

A. gossypii, with its close relationship to Saccharomyces cerevisiae but filamentous growth pattern, provides an interesting evolutionary context for studying DBP10 function. The A. gossypii genome shows high synteny with S. cerevisiae despite their different morphologies and ecological niches, making comparative studies particularly valuable .

How do the biochemical properties of A. gossypii DBP10 compare with homologs from other organisms?

A comparative analysis of DBP10 from different organisms reveals both conserved features and species-specific variations:

Key biochemical properties that may vary include:

• Substrate specificity (preference for specific rRNA structures)

• ATP hydrolysis rates and coupling efficiency

• Thermal stability (reflecting adaptation to preferred growth temperatures)

• Regulation mechanisms (post-translational modifications, protein partners)

• Unwinding or RNP remodeling activity strength

These differences reflect the adaptation of DBP10 to specific cellular environments and ribosome assembly pathways across evolution .

How can DBP10 be used to study co-transcriptional ribosome assembly?

DBP10 provides a valuable tool for investigating co-transcriptional ribosome assembly through several advanced approaches:

• ChIP-seq with DBP10: Chromatin immunoprecipitation followed by sequencing can reveal whether DBP10 associates with ribosomal DNA during transcription, providing evidence for co-transcriptional recruitment.

• Live-cell imaging: Fluorescently tagged DBP10 can be monitored in real-time to visualize its recruitment to nascent pre-ribosomes. This can be combined with MS2-tagged rRNA to simultaneously visualize rRNA transcription and DBP10 recruitment.

• RNA-protein crosslinking: CRAC (crosslinking and analysis of cDNA) or PAR-CLIP methods can identify the precise rRNA binding sites of DBP10 in nascent transcripts.

• Proximity labeling: BioID or APEX2 fused to DBP10 can identify proteins in close proximity during ribosome assembly, revealing the temporal sequence of factor recruitment.

• Conditional depletion systems: Auxin-inducible or other rapid depletion systems targeting DBP10 can reveal immediate consequences on nascent ribosome assembly before secondary effects occur.

These approaches can reveal whether DBP10 functions co-transcriptionally or post-transcriptionally and help construct detailed models of ribosome assembly pathways .

How might DBP10 function be exploited for biotechnological applications in A. gossypii?

Understanding DBP10 function opens several biotechnological possibilities in A. gossypii:

• Enhanced protein production: Modulating DBP10 expression or engineering variants with optimized activity could potentially enhance ribosome biogenesis and thereby increase the protein production capacity of A. gossypii, which is already used for industrial riboflavin production and other biotechnology applications.

• Stress resistance engineering: DEAD-box helicases often function in stress response pathways. Engineering DBP10 or its regulatory networks could potentially enhance A. gossypii resilience to industrial process conditions.

• Synthetic biology applications: Understanding the precise role of DBP10 in coordinating rRNA folding and protein assembly could inform the design of synthetic ribosomes with novel properties.

• Target for antifungal development: Given its essential role, DBP10 could represent a target for developing selective inhibitors against pathogenic fungi while sparing beneficial ones like A. gossypii.

• Metabolic engineering tool: A. gossypii has been successfully engineered to produce various compounds beyond riboflavin, including monoterpenes like sabinene and limonene. Optimizing ribosome biogenesis through DBP10 modulation could potentially enhance these production systems.

These applications would require precise understanding of DBP10's role within the broader context of A. gossypii metabolism and stress responses .

How can multi-omics approaches advance our understanding of DBP10 function?

Integrating multiple omics technologies can provide comprehensive insights into DBP10 function:

By integrating these datasets, researchers can construct comprehensive models of DBP10 function that capture both its direct biochemical activities and broader cellular impacts. This multi-omics approach is particularly powerful when combined with systems biology modeling to predict emergent properties of the system .

What future research directions are most promising for advancing our understanding of DBP10 function?

Several promising research directions could significantly advance our understanding of DBP10:

• Structural studies: Obtaining high-resolution structures of DBP10 alone and in complex with RNA substrates and protein partners would provide mechanistic insights into its function.

• Substrate specificity investigation: Determining the precise RNA structures or sequences that DBP10 recognizes and remodels during ribosome assembly would clarify its specific role.

• In vitro reconstitution: Developing in vitro systems to reconstitute the DBP10-dependent steps of ribosome assembly would allow detailed mechanistic studies.

• Comparative studies: Analyzing DBP10 function across diverse fungal species, including pathogenic and industrially relevant fungi, could reveal evolutionary adaptations and species-specific features.

• Regulatory mechanisms: Investigating how DBP10 activity is regulated during different growth conditions or stress responses could reveal integration with broader cellular signaling networks.

• Development of specific inhibitors: Creating small molecules that specifically inhibit DBP10 would provide valuable research tools and potential leads for antifungal development.

• Single-molecule studies: Applying single-molecule techniques to directly visualize DBP10 activity on RNA substrates would provide unprecedented insights into its mechanism.

These directions would benefit from the application of new technologies and methodological innovations in structural biology, synthetic biology, and single-cell analysis .