Recombinant Debaryomyces hansenii ATP-dependent RNA helicase DBP7 (DBP7), partial

What is DBP7 and what is its primary function in Debaryomyces hansenii?

DBP7 is a putative ATP-dependent RNA helicase belonging to the DEAD-box protein family. Its primary function involves the assembly of pre-ribosomal particles during the biogenesis of the 60S ribosomal subunit. This protein is localized in the nucleolus and plays a crucial role in RNA metabolism, particularly in rRNA processing and ribosome assembly. In D. hansenii specifically, DBP7 contributes to the organism's remarkable stress tolerance capabilities by ensuring proper ribosome biogenesis under high salinity conditions .

How does DBP7 in D. hansenii compare structurally to its homologs in other yeasts like S. cerevisiae?

DBP7 maintains a conserved modular organization across yeast species, comprising a helicase core region with conserved motifs (including the characteristic DEGD motif instead of the DEAD motif found in some related helicases) and variable, non-conserved N- and C-terminal extensions. The helicase core contains RecA-like domains responsible for ATP binding and hydrolysis. While the core region shares high sequence similarity with S. cerevisiae DBP7, the terminal extensions show greater divergence, potentially contributing to D. hansenii's adaptation to high-salt environments. Particularly, the N-terminal domain of D. hansenii DBP7 contains distinct basic residues that may contribute to its function under osmotic stress .

Why is DBP7 considered important for stress adaptation in D. hansenii?

DBP7 contributes to D. hansenii's stress adaptation through its role in ensuring proper ribosome assembly under challenging conditions. When cells experience stress (particularly osmotic or salt stress), maintaining translation efficiency becomes critical. DBP7's function in 60S ribosomal subunit biogenesis ensures that the protein synthesis machinery remains functional despite environmental challenges. The deletion of DBP7 in related yeasts causes severe growth defects, suggesting its importance for cellular fitness, especially under stress conditions. The protein's ability to function in high-salt environments makes it particularly valuable for D. hansenii's halotolerant lifestyle .

What are the key functional domains of DBP7 and how do they contribute to its helicase activity?

DBP7 contains several key domains:

• Helicase Core (amino acids ~163-559): Contains the RecA-like domains with conserved motifs necessary for ATP binding and hydrolysis.

  • Q-motif: Adenine recognition

  • Motif I (DEGD): ATP binding and hydrolysis (Walker A)

  • Motif II: Mg²⁺ coordination (Walker B)

  • Motif III: Coupling ATP hydrolysis to RNA unwinding

  • Motifs IV-VI: RNA binding and helicase activity

• N-terminal Domain (amino acids 1-162): Contains nuclear localization signals and contributes to protein-protein interactions.

• C-terminal Domain (after amino acid 559): Contains a DUF4217 domain of unknown function found in many RNA helicases.

These domains work together to couple ATP hydrolysis with RNA unwinding and remodeling activities .

What is the significance of the N-terminal domain of DBP7, and how can it be experimentally characterized?

The N-terminal domain of DBP7 contains critical features for its function:

• Nuclear Localization Signal (NLS): A basic bipartite NLS within the N-terminal domain (specifically between residues 48-78) is necessary for efficient nuclear import.

• Protein-Protein Interaction Sites: The N-terminus likely mediates interactions with other ribosome assembly factors.

Experimental characterization methods include:

• Truncation Analysis: Creating systematic N-terminal deletions (e.g., ΔN10, ΔN162, ΔNLS) and assessing their impact on localization and function

• Fluorescence Microscopy: Using GFP/YFP fusions to track subcellular localization

• Co-immunoprecipitation: Identifying interaction partners specific to the N-terminal region

• Complementation Assays: Testing the ability of truncated variants to rescue growth defects in DBP7-deleted strains

Results from such experiments in related species show that deletion of the N-terminal domain severely impairs nuclear import and ribosome biogenesis functions .

What role does the C-terminal extension play in DBP7 function and how does it differ from other DEAD-box helicases?

The C-terminal extension of DBP7 contains a DUF4217 domain (Domain of Unknown Function) found in several RNA helicases. Experimental data suggests this domain is essential for DBP7 function:

• Severity of C-terminal Truncations: Deletion of the C-terminal region (e.g., ΔC694-742 and ΔC636-742) impairs cell growth more severely than complete deletion of DBP7, suggesting a dominant-negative effect.

• Pre-ribosomal Particle Association: The C-terminus influences the stable recruitment of DBP7 to pre-60S ribosomal particles.

• Structural Role: It may contribute to the architecture of the RecA-2 domain.

Unlike the C-terminal extensions of spliceosomal RNA helicases (Prp2, Prp16, Prp22, and Prp43), which show sequence conservation among themselves, DBP7's C-terminal extension is unique and likely evolved for its specific function in ribosome biogenesis .

What specific steps of ribosome biogenesis involve DBP7 activity?

DBP7 is involved in early nucleolar stages of 60S ribosomal subunit biogenesis, specifically:

• rRNA Domain Compaction: DBP7 promotes the compaction of domains V and VI of 25S rRNA.

• snoRNA Release: It facilitates the dissociation of snR190 and other snoRNAs from pre-60S particles.

• Scaffolding Complex Dissociation: DBP7 aids in the release of the Npa1 scaffolding complex.

• Ribosomal Protein Recruitment: It enables the recruitment of ribosomal protein uL3, which is critical for forming the peptidyltransferase center (PTC).

• Formation of Mature rRNA: DBP7 is required for the production of 27S and 7S precursor rRNAs, which lead to mature 25S and 5.8S rRNAs.

These steps are sequential and coordinated, with DBP7 acting as a key regulator of early pre-60S maturation events .

How does the absence of DBP7 affect rRNA processing and ribosome assembly?

The absence of DBP7 causes multiple defects in ribosomal RNA processing and assembly:

Notably, the decrease in 27S pre-rRNA is not associated with accumulation of preceding precursors or abnormal intermediates, suggesting rapid degradation of these species in the absence of DBP7 .

What is the relationship between DBP7's ATPase activity and its function in ribosome biogenesis?

DBP7's ATPase activity is essential for its function in ribosome biogenesis:

• ATP-Dependent Remodeling: DBP7 uses ATP hydrolysis to drive conformational changes in RNA structures, particularly in domains V/VI of the 25S rRNA.

• Catalytic Mutant Effects: Mutations in the conserved DEGD motif (e.g., E308Q substitution, creating a DQGD variant) abolish ATPase activity and cause growth defects more severe than the complete absence of DBP7, suggesting a dominant-negative effect.

• ATP Hydrolysis Cycle: The protein binds RNA and ATP, hydrolyzes ATP to ADP + Pi, undergoes conformational change, remodels RNA structure, and then releases RNA and ADP.

• RNA-Dependent ATPase Activity: DBP7's ATPase activity is stimulated by RNA binding, creating a functional coupling between substrate recognition and catalytic activity.

Experimental data from in vitro ATPase assays with recombinant DBP7 confirm that catalytically inactive variants fail to rescue the growth and ribosome assembly defects of DBP7 deletion strains, demonstrating the essential nature of this enzymatic activity .

What are the recommended approaches for recombinant expression and purification of active D. hansenii DBP7?

For successful recombinant expression and purification of active D. hansenii DBP7:

• Expression System:

  • Host: E. coli BL21(DE3) or Rosetta(DE3) for rare codon optimization

  • Vector: pET-based with His10-ZZ tag for affinity purification

  • Induction: 0.5 mM IPTG at lower temperature (18°C) overnight to enhance solubility

• Purification Protocol:

  • Lysis buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 10% glycerol, 2 mM β-mercaptoethanol, protease inhibitors

  • Initial capture: Ni-NTA affinity chromatography

  • Tag removal: TEV protease cleavage

  • Secondary purification: Size exclusion chromatography using Superdex 200 column

  • Storage: 20 mM HEPES pH 7.5, 200 mM NaCl, 10% glycerol, 1 mM DTT at -80°C

• Quality Control Checks:

  • SDS-PAGE for purity assessment

  • Western blotting for identity confirmation

  • ATPase activity assay to confirm functionality

  • Dynamic light scattering for monodispersity

This approach yields approximately 3-5 mg of purified protein per liter of bacterial culture .

How can one assess the ATPase activity of recombinant DBP7 in vitro?

The ATPase activity of recombinant DBP7 can be assessed using several complementary methods:

• Malachite Green Phosphate Assay:

  • Principle: Measures free inorganic phosphate released during ATP hydrolysis

  • Reaction conditions: 2-5 μM DBP7, 1 mM ATP, 2 mM MgCl₂, 50 mM KCl, 20 mM HEPES pH 7.5

  • RNA dependence: Include 0-100 μg/ml total RNA or specific RNA substrates

  • Controls: Include catalytically inactive DBP7 (DQGD mutant)

  • Detection: Measure absorbance at 620 nm after Malachite Green addition

• Thin-Layer Chromatography (TLC) with [γ-³²P]-ATP:

  • Principle: Separates radioactive ATP from released phosphate

  • Reaction conditions: Similar to above but with [γ-³²P]-ATP tracer

  • Separation: PEI-cellulose TLC plates, 0.75 M KH₂PO₄ as mobile phase

  • Detection: Phosphorimager or autoradiography

  • Quantification: Calculate percentage of hydrolyzed ATP

• Coupled Enzyme Assay:

  • Principle: Couples ATP hydrolysis to NADH oxidation via pyruvate kinase and lactate dehydrogenase

  • Detection: Continuous monitoring of NADH absorption decrease at 340 nm

  • Advantages: Real-time kinetic measurements

Expected parameters for wild-type DBP7 include an RNA-stimulated ATPase rate of approximately 1-3 ATP molecules per minute per enzyme molecule at 30°C .

What methods can be used to investigate the interaction between DBP7 and pre-ribosomal particles?

Multiple complementary approaches can investigate DBP7-pre-ribosomal particle interactions:

• Sucrose Density Gradient Analysis:

  • Prepare cell lysates under non-denaturing conditions

  • Separate complexes by centrifugation through 10-50% sucrose gradients

  • Collect fractions and analyze by:

    • Absorbance profiling at 260 nm (ribosomal peaks)

    • Western blotting (DBP7 detection)

    • Northern blotting (rRNA species)

  • Expected result: DBP7 co-sediments with pre-60S fractions

• Co-Immunoprecipitation (Co-IP):

  • Generate epitope-tagged DBP7 (e.g., HA-DBP7, DBP7-TAP)

  • Immunoprecipitate using tag-specific antibodies

  • Analyze precipitated RNAs by:

    • Northern blotting (specific rRNAs, snoRNAs)

    • RNA-seq (unbiased approach)

  • Analyze co-precipitated proteins by:

    • Western blotting (targeted approach)

    • Mass spectrometry (proteomics approach)

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

  • UV-crosslink RNA-protein complexes in vivo

  • Purify DBP7-RNA complexes under denaturing conditions

  • Sequence associated RNAs to map binding sites

  • Expected targets: domains V/VI of 25S rRNA, near snR190 binding site

• Structure Determination Methods:

  • Cryo-EM of DBP7-bound pre-60S particles

  • X-ray crystallography of DBP7 with RNA fragments

  • Integrative modeling approaches

These methods have revealed that DBP7 associates with early nucleolar pre-60S particles and binds specifically to domains V/VI of the 25S rRNA .

What advantages does D. hansenii offer as a model organism for studying DBP7 compared to S. cerevisiae?

D. hansenii offers several distinct advantages for DBP7 research:

• Stress Tolerance Context:

  • Extreme halotolerance (growth in up to 4M NaCl)

  • Adaptation to osmotic stress

  • Xerotolerance (drought resistance)

  • Growth at extreme pH and temperature ranges
    These properties allow studying DBP7 function under stress conditions relevant to biotechnological applications.

• Evolutionary Perspective:

  • Distinct evolutionary history from S. cerevisiae

  • Different codon usage and gene regulation patterns

  • Variations in ribosome structure and assembly
    These differences provide comparative insights into DBP7 function conservation.

• Biotechnological Applications:

  • Can grow in industrial by-products with high salt content

  • Oleaginous nature (accumulates lipids)

  • Probiotic properties
    These features make findings potentially applicable to industrial contexts.

• Genetic Manipulation Tools:

  • Recently developed CRISPR-Cas9 systems adapted for D. hansenii

  • PCR-based gene targeting with high efficiency (>75%)

  • In vivo DNA assembly capabilities
    These tools enable sophisticated genetic studies previously limited to conventional yeasts .

What are the current genetic tools available for manipulating DBP7 in D. hansenii?

A suite of advanced genetic tools is now available for DBP7 manipulation in D. hansenii:

• PCR-based Gene Targeting:

  • High-efficiency homologous recombination (>75%) using 50 bp flanking homology

  • Wild-type isolates can be directly transformed without prior auxotrophic marker creation

  • Heterologous selectable markers function effectively

• CRISPR-CUG/Cas9 System:

  • Adapted specifically for D. hansenii's alternative genetic code (CUG→Ser instead of Leu)

  • Enables precise genome editing without extensive homology arms

  • Can create point mutations, deletions, and insertions

• In Vivo DNA Assembly:

  • Co-transformation of multiple DNA fragments with 30 bp homologous overlaps

  • Allows assembly of up to three fragments in correct order in a single step

  • Useful for promoter-gene-terminator construct creation

• Expression Optimization Tools:

  • High-efficiency promoters (TEF1 from Arxula adeninivorans)

  • Effective terminators (CYC1 from S. cerevisiae)

  • Functional secretion signals (α-MF from S. cerevisiae)

These tools enable sophisticated molecular genetic analyses of DBP7, including creation of point mutations, domain deletions, promoter swaps, and fluorescent protein fusions .

How can researchers address the challenges of working with D. hansenii's alternative genetic code when expressing recombinant DBP7?

D. hansenii uses an alternative genetic code where the CUG codon encodes serine instead of the standard leucine, creating challenges for recombinant expression. Researchers can address these issues through:

• Codon Optimization Strategies:

  • Avoid CUG codons in heterologous expression constructs

  • Replace CUG codons with other serine codons (UCN) in genes to be expressed in D. hansenii

  • Use codon optimization software with D. hansenii-specific settings

  • For DBP7 expression in E. coli, replace D. hansenii CUG-encoded serines with leucines if they are not functionally critical

• Expression Systems Considerations:

  • For D. hansenii native expression: no codon adaptation needed

  • For heterologous proteins in D. hansenii: avoid CUG codons or replace with UCN

  • For D. hansenii proteins in E. coli: consider the impact of CUG→Ser substitutions on structure/function

• Verification Methods:

  • Sequence verification of all constructs

  • Mass spectrometry to confirm correct amino acid incorporation

  • Functional assays to ensure protein activity

• Use of Specialized Expression Strains:

  • When expressing in E. coli, rare codon optimization strains (e.g., Rosetta) can help with other rare codons, though they don't address the CUG issue

These approaches ensure that recombinant DBP7 maintains its native sequence and functionality despite the alternative genetic code challenges .

How does DBP7 coordinate with other RNA helicases in ribosome biogenesis, and what techniques can resolve their temporal and functional relationships?

DBP7 functions within a network of at least 20 RNA helicases involved in ribosome biogenesis. Understanding their coordination requires sophisticated approaches:

• Sequential Action Analysis:

  • Depletion experiments with auxin-inducible degron-tagged helicases

  • Pulse-chase labeling of rRNA combined with helicase depletion

  • Quantitative mass spectrometry of pre-ribosomal intermediates

• Functional Redundancy Assessment:

  • Synthetic genetic interaction screens between helicase mutants

  • Double conditional mutants with varying depletion timing

  • Suppressor screens to identify bypass pathways

• Physical Interaction Mapping:

  • Proximity labeling (BioID or APEX) with DBP7 as bait

  • Cryo-EM of sequential pre-ribosomal particles

  • Single-molecule fluorescence microscopy with differentially labeled helicases

• Substrate Specificity Determination:

  • CRAC or similar crosslinking methods applied to multiple helicases

  • In vitro competition assays for RNA binding sites

  • Structure determination of sequential remodeling events

Current data suggest DBP7 acts early in 60S biogenesis, potentially coordinating with Dbp9 and Drs1, which act in similar regions of the pre-rRNA .

What methodological approaches can resolve contradictory findings about DBP7's influence on rRNA 2'-O-methylation patterns?

Contradictory findings regarding DBP7's effect on rRNA 2'-O-methylation can be resolved through:

• High-Resolution Methylation Analysis:

  • RiboMeth-seq (RMS) with increased sequencing depth

  • Site-specific 2'-O-methylation quantification using reverse transcription-based methods

  • Mass spectrometry of individual nucleotides

• Temporal Dynamics Assessment:

  • Time-course experiments during DBP7 depletion

  • Pulse-chase labeling combined with methylation analysis

  • Single-molecule approaches to track methylation timing

• Mechanistic Dissection:

  • Analysis of snoRNA association with pre-ribosomes in DBP7 mutants

  • In vitro reconstitution of methylation reactions with/without DBP7

  • Structural studies of DBP7 interaction with methylation machinery

• Quantitative Controls:

  • Precise quantification of methylation levels

  • Statistical approaches to distinguish biological from technical variability

  • Multiple biological replicates

Published data indicates mild but significant changes in the 2'-O-methylation of specific 25S rRNA nucleotides (C663, U898, C2197, U2347, A2640, and C2948) in the absence of DBP7, with most sites showing decreased methylation .

How might the mechanistic insights from DBP7 research be applied to understand RNA helicase dysfunction in human ribosomopathies?

The mechanistic insights from DBP7 research have significant implications for understanding human ribosomopathies:

• Translational Research Opportunities:

  • Human homologs of yeast RNA helicases implicated in ribosomopathies

  • Conserved structural features and functional mechanisms

  • Similar coordination of rRNA folding and ribosomal protein incorporation

• Methodological Approaches:

  • CRISPR-Cas9 editing of helicase domains in human cells

  • Patient-derived cells carrying helicase mutations

  • Humanized yeast models expressing human helicase variants

  • Cryo-EM structures of disease-relevant pre-ribosomal intermediates

• Therapeutic Target Identification:

  • Small molecule screens for compounds that rescue RNA helicase mutant phenotypes

  • Structure-based drug design targeting specific conformational states

  • Genetic suppressor screens to identify bypass pathways

• Computational Modeling:

  • Molecular dynamics simulations of mutant helicases

  • Systems biology approaches to model ribosome assembly networks

  • Integration of genomic, structural, and functional data

RNA helicase dysfunction is implicated in multiple ribosomopathies including Diamond-Blackfan anemia and Shwachman-Diamond syndrome, making mechanistic insights from model systems like yeast DBP7 particularly valuable for understanding disease pathogenesis .

How can DBP7's function in D. hansenii be exploited for biotechnological applications in high-salt environments?

DBP7's role in D. hansenii can be leveraged for several biotechnological applications:

• Engineered Stress Tolerance:

  • Overexpression of DBP7 may enhance ribosome production under stress

  • Engineering DBP7 variants with improved ATPase activity in high salt

  • Using DBP7 promoter/regulatory elements for salt-inducible expression systems

• Recombinant Protein Production:

  • Development of D. hansenii strains with optimized DBP7 function for improved translation in high-salt media

  • Creation of robust expression systems that maintain protein synthesis during osmotic stress

  • Engineering of stable ribosome populations for industrial fermentation

• Bioprocess Applications:

  • Non-sterile fermentations in high-salt media where DBP7 function supports D. hansenii growth

  • Utilization of salt-rich industrial by-products as growth media

  • Development of continuous fermentation processes in challenging environments

• Synthetic Biology Platforms:

  • Creation of salt-inducible genetic circuits using DBP7 regulatory elements

  • Development of riboswitch-based controls linked to DBP7 function

  • Engineering of synthetic ribosome assembly pathways with modified DBP7

These applications leverage D. hansenii's natural halotolerance and DBP7's role in maintaining translation under stress conditions .

What techniques can be used to investigate the potential interplay between DBP7 function and D. hansenii's remarkable osmotolerance?

To investigate links between DBP7 and osmotolerance:

• Functional Genomics Approaches:

  • Transcriptomics comparing wild-type and dbp7Δ strains across salt gradients

  • Ribosome profiling to assess translation efficiency under osmotic stress

  • Phosphoproteomics to identify stress-responsive signaling affecting DBP7

• Genetic Interaction Mapping:

  • Synthetic genetic array analysis crossing dbp7 mutants with HOG pathway components

  • CRISPR interference screens in osmotic stress conditions

  • Multicopy suppressor screens to identify genes that compensate for DBP7 deficiency

• In Vivo Functional Assays:

  • FRET biosensors to monitor DBP7 conformational changes during osmotic shifts

  • Single-molecule tracking of fluorescently labeled DBP7 during stress response

  • Selective ribosome profiling of DBP7-associated ribosomes

• Structural Studies:

  • Cryo-EM of ribosomes from salt-stressed cells with/without DBP7

  • Hydrogen-deuterium exchange mass spectrometry to detect conformational changes

  • In vitro reconstitution of ribosome assembly under varying ionic conditions

These approaches can reveal how DBP7's function in ribosome biogenesis contributes to D. hansenii's ability to thrive in high-salt environments .

What are the emerging techniques for studying the dynamics of DBP7-mediated RNA remodeling during ribosome assembly?

Cutting-edge techniques for studying DBP7-mediated RNA remodeling include:

• Single-Molecule Approaches:

  • Single-molecule FRET to monitor RNA conformational changes in real-time

  • Optical tweezers to measure forces during RNA unwinding

  • Zero-mode waveguides for observing DBP7-RNA interactions at physiological concentrations

• Time-Resolved Structural Methods:

  • Time-resolved cryo-EM with millisecond freezing after ATP addition

  • Chemical footprinting with high temporal resolution

  • Hydrogen-deuterium exchange mass spectrometry with quench-flow apparatus

• In-Cell Probing Technologies:

  • DMS-MaPseq for in vivo RNA structure mapping

  • SHAPE-MaP for nucleotide-resolution RNA structure determination

  • Selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP)

• Correlative Imaging Approaches:

  • Correlative light and electron microscopy (CLEM) of labeled DBP7

  • Super-resolution microscopy combined with expansion microscopy

  • Live-cell single-particle tracking of DBP7 dynamics

These methods can capture the transient states and dynamic rearrangements mediated by DBP7 during ribosome assembly, providing unprecedented insights into the molecular mechanisms of RNA helicase function .