Recombinant Neurospora crassa ATP-dependent RNA helicase dbp-7 (dbp-7), partial

What is Neurospora crassa ATP-dependent RNA helicase dbp-7?

Neurospora crassa ATP-dependent RNA helicase dbp-7 belongs to the DEAD-box protein family of RNA helicases that utilize ATP hydrolysis to unwind RNA secondary structures. Similar to its yeast homolog, it likely plays critical roles in ribosome biogenesis, particularly in the assembly of the large ribosomal subunit (LSU). The protein contains characteristic conserved motifs including the DEAD (Asp-Glu-Ala-Asp) sequence that is essential for its ATP-dependent helicase activity . Understanding its function has been advanced through comparative studies with homologs in other organisms, particularly the well-characterized Dbp7 in yeast.

How does dbp-7 function compare to homologs in other organisms?

The function of Neurospora crassa dbp-7 can be inferred from studies of homologs in other organisms. In yeast, Dbp7 binds specifically to domain V/VI of early pre-60S ribosomal particles and is critical for multiple aspects of large subunit ribosome biogenesis . Its absence impairs dissociation of the Npa1 scaffolding complex, release of the snR190 folding chaperone, recruitment of A3 cluster factors, and binding of the ribosomal protein uL3 . While the DBP gene in mice has been shown to function in circadian rhythm regulation , the specific role of Neurospora crassa dbp-7 in circadian processes remains to be fully elucidated, though Neurospora is a well-established model organism for studying circadian rhythms.

What are the structural domains of dbp-7 and their functions?

Neurospora crassa dbp-7, like other DEAD-box helicases, likely contains two RecA-like domains connected by a flexible linker. These domains work together to bind RNA and ATP. The N-terminal domain typically contains motifs I, Ia, Ib, II (DEAD), and III, which are primarily involved in ATP binding and hydrolysis. The C-terminal domain contains motifs IV, V, and VI, which participate in RNA binding and communication between the ATP and RNA binding sites. The specificity of dbp-7 for its target RNAs is likely determined by additional sequences outside these conserved motifs. In yeast Dbp7, the protein binds specifically to domain V/VI of pre-60S ribosomal particles, which contains the peptidyltransferase center (PTC) .

What expression systems are most effective for producing recombinant Neurospora crassa dbp-7?

For expression of recombinant Neurospora crassa dbp-7, several host systems can be employed including E. coli, yeast, baculovirus, or mammalian cell systems . Each system offers advantages depending on research requirements:

For functional studies requiring authentic post-translational modifications, yeast expression systems may be preferable given that dbp-7 is a fungal protein. E. coli systems are advantageous for structural studies requiring large quantities of protein.

What purification strategy yields the highest purity for recombinant dbp-7?

A multi-step purification strategy is recommended for obtaining high-purity recombinant dbp-7. Standard protocols can achieve ≥85% purity as determined by SDS-PAGE . An effective purification workflow includes:

• Affinity chromatography using His-tag or GST-tag (primary capture)

• Ion exchange chromatography (intermediate purification)

• Size exclusion chromatography (polishing step)

For enhanced purity, consider incorporating these additional steps:

• ATP-agarose affinity chromatography to select for functional protein

• Heparin affinity chromatography (mimics RNA interaction)

• Hydrophobic interaction chromatography

Include protease inhibitors and maintain low temperatures throughout purification to minimize degradation. For highest activity, include ATP and magnesium in storage buffers, and assess final purity using both SDS-PAGE and activity assays.

How should researchers assess the activity of purified recombinant dbp-7?

Assessment of recombinant dbp-7 activity should include both ATPase and RNA unwinding assays:

ATPase Activity Assay:

• Measure ATP hydrolysis using a malachite green phosphate detection system

• Compare rates with and without RNA substrates (RNA-dependent ATPase activity)

• Determine kinetic parameters (Km, Vmax) using varying ATP concentrations

RNA Unwinding Assay:

• Design RNA duplexes with partial complementarity and fluorescent labels

• Monitor unwinding activity through fluorescence changes in real-time

• Assess ATP-dependence by performing assays with non-hydrolyzable ATP analogs

Complementary methods include thermal stability assays (TSA) to verify proper folding and RNA binding assays using electrophoretic mobility shift assays (EMSA) or surface plasmon resonance (SPR) to confirm target RNA interaction.

How can researchers use recombinant dbp-7 to study ribosome biogenesis?

Recombinant dbp-7 can be employed to investigate ribosome biogenesis through several experimental approaches:

• Reconstitution Assays: Use purified dbp-7 in in vitro reconstitution of pre-ribosomal complexes to assess its direct effect on rRNA folding and compaction, particularly in domain V/VI regions.

• RNA Structural Analysis: Employ techniques such as SHAPE (Selective 2'-Hydroxyl Acylation analyzed by Primer Extension) or DMS-MaPseq to analyze RNA structural changes induced by dbp-7 activity.

• Pull-down Experiments: Utilize tagged recombinant dbp-7 to identify interacting partners in ribosome assembly, similar to studies in yeast showing Dbp7 interactions with the Npa1 complex and A3 cluster factors .

• In vitro Translation Assays: Assess the functional consequences of dbp-7 activity by measuring translation efficiency of ribosomes assembled with or without active dbp-7.

• Cryo-EM Analysis: Use recombinant dbp-7 to stabilize specific pre-ribosomal complexes for structural determination, providing insights into the remodeling events catalyzed by this helicase.

A combined approach using these techniques could reveal how dbp-7 contributes to the formation of functional ribosomes in Neurospora crassa.

What protocols are recommended for studying dbp-7's potential role in circadian rhythm regulation?

To investigate dbp-7's potential role in circadian rhythm regulation in Neurospora crassa, researchers should consider these methodological approaches:

• Temporal Expression Analysis: Perform time-course experiments using RT-qPCR to analyze dbp-7 mRNA expression over circadian time (similar to analyses performed for DBP in mice ).

• In situ Hybridization: Examine tissue-specific expression patterns across the circadian cycle, focusing on regions equivalent to the SCN in mice where DBP shows strong rhythmic expression .

• Knockout/Knockdown Studies: Generate dbp-7 knockout or knockdown strains and analyze circadian phenotypes using race tube assays (standard for Neurospora circadian studies).

• ChIP-Seq Analysis: Identify potential clock-controlled genes regulated by dbp-7 through chromatin immunoprecipitation followed by sequencing.

• Protein-Protein Interaction Studies: Investigate interactions between dbp-7 and known circadian clock components (WCC, FRQ, FRH) using co-immunoprecipitation or yeast two-hybrid assays.

These approaches can be integrated to determine whether dbp-7 displays rhythmic expression similar to mouse DBP and to elucidate its potential roles in the Neurospora circadian system.

What RNA substrates should be used to characterize dbp-7's helicase activity?

When characterizing dbp-7's helicase activity, substrate selection should be guided by its presumed biological function. Based on homology to yeast Dbp7, which interacts with domain V/VI of pre-rRNA , the following RNA substrates are recommended:

• Pre-rRNA-derived Substrates:

  • Synthetic RNA oligonucleotides corresponding to domain V/VI of Neurospora 25S rRNA

  • Duplex RNAs mimicking native secondary structures in these regions

  • Full-length in vitro transcribed pre-rRNA sections containing the peptidyltransferase center

• Generic Helicase Substrates:

  • RNA duplexes with varying stability (GC content)

  • Substrates with different overhang configurations (5', 3', or blunt ends)

  • RNA/DNA hybrid substrates to test specificity

• Specialty Substrates:

  • Substrates with RNA modifications found in native rRNA

  • RNA structures known to be difficult to resolve (G-quadruplexes, triple helices)

  • Substrates designed to test directionality of unwinding

A comprehensive analysis should include kinetic parameters for each substrate type, allowing comparison of dbp-7's activity across different RNA contexts.

How does dbp-7 coordinate with other helicases and RNA-binding proteins during ribosome assembly?

Based on studies of the yeast homolog, dbp-7 likely functions within a complex network of proteins during ribosome assembly. To study these interactions:

• Sequential Helicase Activity Assays: Design experiments to determine the order of action between dbp-7 and other helicases known to function in ribosome biogenesis. In yeast, Dbp7 acts early in pre-60S ribosomal particle maturation .

• Co-factor Requirements Analysis: Determine whether dbp-7 requires specific protein co-factors for optimal activity, similar to how many DEAD-box proteins function in complexes rather than in isolation.

• Reconstitution of Minimal Functional Units: Systematically assemble combinations of helicases and binding proteins to determine the minimal set required for specific rRNA remodeling events.

• Single-molecule FRET Experiments: Visualize the sequential binding and activity of different factors during ribosome assembly, with particular focus on domain V/VI where yeast Dbp7 binds .

• Cryo-EM Structural Studies: Capture transition states during the assembly process to visualize how dbp-7 and other factors coordinate structural changes.

Understanding these coordination events is crucial for developing a comprehensive model of ribosome assembly.

What are the molecular mechanisms underlying the potential circadian functions of dbp-7?

While direct evidence for dbp-7's role in Neurospora circadian rhythms is limited, the functions of its homolog in mice provide a framework for investigation . To elucidate the molecular mechanisms:

• Promoter Analysis: Examine the dbp-7 promoter for binding sites of known circadian transcription factors, particularly the White Collar Complex (WCC).

• Transcriptional Regulation Studies: Determine whether dbp-7 expression is directly regulated by core clock components using chromatin immunoprecipitation (ChIP) assays.

• Protein Stability and Modification Analysis: Investigate whether dbp-7 protein undergoes rhythmic post-translational modifications or degradation, contributing to its potential rhythmic activity.

• Target Identification: Perform RNA immunoprecipitation followed by sequencing (RIP-seq) to identify RNAs bound by dbp-7 at different circadian times.

• Comparative Analysis with DBP: Create chimeric proteins combining domains from Neurospora dbp-7 and mouse DBP to identify functionally conserved regions important for circadian function .

These approaches can reveal whether dbp-7 functions similarly to mouse DBP, which influences circadian period length and activity levels .

How do mutations in ATP-binding motifs affect dbp-7's function in ribosome biogenesis?

Structure-function analysis of dbp-7 can be performed by creating specific mutations in its conserved motifs:

For each mutation, researchers should:

• Express and purify the mutant protein

• Characterize its biochemical properties (ATP binding/hydrolysis, RNA binding/unwinding)

• Assess its ability to complement dbp-7 deletion in vivo

• Examine effects on pre-rRNA processing and ribosome assembly

This approach has been valuable in understanding yeast Dbp7, where catalytic activity is essential for pre-60S maturation and preventing degradation of early pre-ribosomal particles .

What are common challenges in working with recombinant dbp-7 and how can they be addressed?

Researchers frequently encounter several challenges when working with recombinant dbp-7:

• Protein Solubility Issues:

  • Problem: Formation of inclusion bodies in E. coli

  • Solution: Express at lower temperatures (16-18°C), use solubility-enhancing tags (MBP, SUMO), or switch to eukaryotic expression systems

• Low ATPase Activity:

  • Problem: Purified protein shows minimal ATP hydrolysis

  • Solution: Ensure presence of divalent cations (Mg²⁺), test different RNA cofactors, verify protein folding using circular dichroism

• RNA Substrate Specificity:

  • Problem: Difficulty identifying physiologically relevant substrates

  • Solution: Use SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to identify preferred binding sequences, or focus on rRNA segments from domain V/VI based on yeast Dbp7 specificity

• Protein Stability During Storage:

  • Problem: Activity loss during storage

  • Solution: Store with 10-20% glycerol, include reducing agents, avoid freeze-thaw cycles, consider flash-freezing aliquots in liquid nitrogen

• Assay Interference:

  • Problem: Contaminating nucleases or phosphatases affecting results

  • Solution: Include EDTA in storage buffers (remove before assays requiring Mg²⁺), use ultra-pure reagents, include additional purification steps

Implementing these solutions can significantly improve experimental outcomes when working with this challenging but important RNA helicase.

How can researchers effectively study the interaction between dbp-7 and pre-ribosomal complexes?

To study interactions between dbp-7 and pre-ribosomal complexes, researchers should employ multiple complementary approaches:

• Gradient Analysis of Pre-ribosomes:

  • Fractionate cell lysates on sucrose gradients and analyze dbp-7 co-sedimentation with pre-ribosomal particles

  • Compare wild-type distribution with catalytically inactive mutants

• CRAC (UV Cross-linking and Analysis of cDNA):

  • Identify precise RNA binding sites by UV-crosslinking tagged dbp-7 to associated RNAs

  • Sequence recovered RNAs to map binding sites at nucleotide resolution

• Proximity Labeling Approaches:

  • Use BioID or APEX2 fused to dbp-7 to identify proteins in close proximity within pre-ribosomal complexes

  • Compare results at different stages of ribosome maturation

• Fluorescence Microscopy:

  • Visualize co-localization of fluorescently tagged dbp-7 with pre-ribosomal markers

  • Employ FRAP (Fluorescence Recovery After Photobleaching) to assess dynamics of association

• Reconstituted Systems:

  • Use purified components to reconstitute minimal pre-ribosomal complexes in vitro

  • Assess binding affinities and structural changes upon dbp-7 association

These techniques, particularly when used in combination, can provide comprehensive insights into how dbp-7 interacts with and remodels pre-ribosomal complexes similar to the functions observed for yeast Dbp7 .

What approaches are most effective for analyzing dbp-7's impact on rRNA structure?

To analyze dbp-7's impact on rRNA structural changes, researchers should consider these state-of-the-art methodologies:

• Chemical Probing Methods:

  • SHAPE (Selective 2'-Hydroxyl Acylation analyzed by Primer Extension) to monitor RNA flexibility changes

  • DMS-MaPseq for determining single-strand regions in vivo

  • PARIS (Psoralen Analysis of RNA Interactions and Structures) to capture RNA duplexes

• Cryo-EM Analysis:

  • Compare structures of pre-ribosomal particles with and without dbp-7

  • Use catalytically inactive mutants to trap transition states

  • Focus on domain V/VI structure, where yeast Dbp7 functions

• FRET-based Assays:

  • Design fluorescently labeled RNA constructs to monitor conformational changes

  • Perform single-molecule FRET to observe transient intermediates during unwinding

• Hydroxyl Radical Footprinting:

  • Map RNA regions protected by dbp-7 binding

  • Identify structural changes in the presence of ATP vs. non-hydrolyzable analogs

• RNA-Seq Analysis:

  • Compare pre-rRNA processing patterns in wild-type vs. dbp-7 mutants

  • Identify processing intermediates that accumulate in the absence of functional dbp-7

These approaches, particularly when combined, can reveal how dbp-7 structurally remodels rRNA during ribosome biogenesis, similar to the domain V/VI compaction function demonstrated for yeast Dbp7 .