Recombinant Neurospora crassa Transcription activator of gluconeogenesis NCU03938 (G17A4.270, NCU03938), partial

What is the NCU03938 protein and what is its role in Neurospora crassa?

NCU03938 (also known as G17A4.270) is a transcription activator involved in gluconeogenesis regulation in the filamentous fungus Neurospora crassa. It belongs to the zinc-cluster family of transcription factors, containing a DNA-binding domain characteristic of these proteins. The full-length native protein consists of 740 amino acid residues and functions as a regulator of metabolic pathways, particularly in carbon metabolism . The protein plays a crucial role in coordinating gene expression during glucose limitation, activating genes involved in the synthesis of glucose from non-carbohydrate carbon sources. As a zinc-cluster transcription factor, it recognizes specific DNA sequences in the promoter regions of target genes, allowing N. crassa to adapt to changing environmental carbon availability .

How should the recombinant NCU03938 protein be stored and handled in laboratory settings?

For optimal stability and activity of recombinant NCU03938, proper storage conditions are essential. The lyophilized form maintains stability for approximately 12 months when stored at -20°C/-80°C, while the reconstituted liquid form is stable for about 6 months at the same temperatures . For working aliquots, storage at 4°C is recommended for up to one week .

Importantly, repeated freezing and thawing cycles should be avoided as they can compromise protein integrity and activity . For reconstitution, the manufacturer recommends:

• Brief centrifugation of the vial before opening

• Reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Addition of glycerol to a final concentration of 5-50% (50% being the standard recommendation) for long-term storage

• Aliquoting the solution to minimize freeze-thaw cycles

What are the structural characteristics of the NCU03938 transcription factor?

The NCU03938 protein is characterized as a zinc-cluster transcription factor, a family of proteins that typically contain a Zn(II)2Cys6 binuclear cluster domain that mediates DNA binding. While the full-length protein is 740 amino acids, commercial recombinant versions are often provided as partial proteins focusing on functional domains .

Based on similar zinc-cluster transcription factors studied in N. crassa, NCU03938 likely contains:

• An N-terminal DNA-binding domain with conserved cysteine residues that coordinate zinc ions

• A middle homology region that mediates dimerization

• A C-terminal activation domain involved in transcriptional activation

For experimental applications requiring just the DNA-binding activity, expression constructs containing the first 426 base pairs of the coding sequence have been successfully used for similar zinc-cluster transcription factors in N. crassa .

What expression systems are recommended for producing functional recombinant NCU03938?

Based on established protocols for similar N. crassa transcription factors, E. coli-based expression systems are effective for producing recombinant NCU03938 . The commercially available recombinant protein is produced in E. coli with purity greater than 85% as verified by SDS-PAGE .

For researchers developing their own expression systems, the following methodology has proven successful for similar zinc-cluster transcription factors from N. crassa:

• Clone the coding sequence of interest (full-length or domain-specific) into a suitable expression vector such as pET-26b

• Incorporate restriction sites (e.g., NdeI and XhoI) for directional cloning

• Include a C-terminal hexahistidine tag for purification purposes

• Transform into an expression strain like Rosetta (DE3) cells that can account for codon usage differences

• For optimal expression of zinc-cluster proteins, supplement growth media with ZnCl₂

• Use lysozyme treatment and sonication for cell lysis and protein extraction

When expressing only the DNA-binding domain, researchers have successfully amplified the first 426 bp of the coding sequence for similar N. crassa transcription factors .

What purification methods yield the highest purity and activity for recombinant NCU03938?

For optimal purification of recombinant NCU03938 with maintained biological activity, a multi-step purification process is recommended:

• Initial Clarification: Following cell lysis via sonication or enzymatic methods, centrifugation at high speed (>15,000 × g) for 30 minutes to remove cellular debris

• Affinity Chromatography: For His-tagged constructs, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin with step-wise imidazole elution (50-300 mM)

• Ion Exchange Chromatography: As a secondary purification step to remove contaminants and truncated forms

• Size Exclusion Chromatography: For final polishing and buffer exchange

• Quality Control: Verification of purity using SDS-PAGE (>85% purity should be achieved)

Throughout the purification process, it's critical to maintain conditions that preserve zinc binding, including the presence of reducing agents like DTT to prevent oxidation of cysteine residues essential for zinc coordination .

What are the optimal conditions for assessing NCU03938 DNA-binding activity?

To assess the DNA-binding activity of NCU03938, Electrophoretic Mobility Shift Assay (EMSA) has been successfully employed for similar zinc-cluster transcription factors in N. crassa. Based on established protocols, the following conditions are recommended:

• Probe Preparation:

  • Generate double-stranded DNA oligonucleotides containing putative binding sites

  • Include seven-nucleotide overhangs for radiolabeling

  • Hybridize complementary oligonucleotides (20 nmol each) in annealing buffer (10 mM Tris-HCl pH 7.9, 50 mM NaCl, 10 mM MgCl₂, 1 mM DTT)

  • Fill in 5' overhangs using Klenow enzyme with radiolabeled [α-³²P]dCTP (3000 Ci/mmol)

• Binding Reaction:

  • Include purified recombinant protein in a buffer containing zinc

  • Maintain reducing conditions to preserve zinc coordination

  • Include appropriate controls (empty vector expressed protein)

• Electrophoresis Conditions:

  • Use non-denaturing polyacrylamide gels

  • Include mobility controls to verify specific binding

For characterizing new binding sites, both wild-type and mutant oligonucleotides should be employed to confirm sequence specificity .

How does NCU03938 integrate into the broader transcriptional regulatory network of Neurospora crassa?

NCU03938 functions within a complex transcriptional regulatory network in N. crassa, particularly in relation to metabolic adaptation and gluconeogenesis. Transcription factor interactions in N. crassa have been extensively curated, revealing important insights about network structure:

• Network Connectivity: High-confidence transcriptional regulatory networks in N. crassa involve 58 transcription factors and 602 TF-target interactions .

• Evidence Classification: Regulatory interactions are supported by two types of evidence:

  • "Binding" evidence (ChIP, EMSA, DNA footprinting)

  • "TF perturbation" evidence (differential expression after TF modification)

  • In N. crassa, 704 interactions are supported by both types of evidence

• Combinatorial Regulation: Many target genes in N. crassa are regulated by multiple transcription factors, creating complex regulatory circuits. This suggests NCU03938 likely functions in concert with other factors to fine-tune gluconeogenesis .

• Autoregulation: Several N. crassa transcription factors exhibit autoregulation, either activating or repressing their own expression. For example, WC-1, CPC-1, ACR-2, QA-1F, and FL activate their own expression. While not specifically documented for NCU03938, this regulatory mechanism should be investigated .

Understanding NCU03938's position within this network is crucial for comprehending its role in metabolic adaptation, particularly during carbon source switching and stress responses.

What experimental approaches can identify genome-wide binding sites for NCU03938?

For comprehensive identification of NCU03938 binding sites across the N. crassa genome, several complementary approaches can be employed:

• ChIP-seq Analysis:

  • Crosslink protein-DNA complexes in vivo

  • Immunoprecipitate using antibodies against NCU03938 or epitope tags

  • Sequence recovered DNA fragments

  • Map to the N. crassa genome to identify binding regions

  • This high-throughput approach has successfully identified numerous interactions in N. crassa

• DNA Affinity Purification sequencing (DAP-seq):

  • Incubate purified recombinant NCU03938 with fragmented genomic DNA

  • Purify protein-bound DNA fragments

  • Sequence and map to the genome

  • Provides in vitro binding profile without cellular context

• Motif Analysis:

  • Use sequences identified from ChIP-seq or DAP-seq to derive consensus binding motifs

  • Apply computational approaches to predict additional binding sites throughout the genome

  • Validate high-confidence predictions experimentally

• Validation Experiments:

  • Confirm direct binding using EMSAs for selected targets

  • Assess functional consequences through reporter gene assays

  • Evaluate target gene expression changes in NCU03938 mutant strains

The integration of high-throughput and low-throughput methods provides the most comprehensive understanding, as only 2.9% of N. crassa transcriptional interactions are supported by low-throughput studies, despite their higher confidence .

What is the relationship between NCU03938 and other zinc-cluster transcription factors in regulating metabolic pathways?

Zinc-cluster transcription factors in N. crassa, including NCU03938, often exhibit functional relationships in the regulation of metabolic pathways. This coordination ensures appropriate cellular responses to environmental conditions. Analysis of regulatory networks reveals:

• Co-regulatory Patterns: In N. crassa, multiple zinc-cluster transcription factors can regulate the same target genes. The seven most highly regulated targets in N. crassa encode glycoside hydrolases, suggesting coordination of carbohydrate metabolism genes by multiple factors .

• Hierarchical Organization: Some transcription factors regulate other transcription factors, creating regulatory cascades. For NCU03938, its position in this hierarchy can be determined through network analysis of ChIP-seq and gene expression data .

• Metabolic Specialization: Different zinc-cluster transcription factors often control distinct yet interconnected metabolic pathways. While NCU03938 focuses on gluconeogenesis, it likely interfaces with factors controlling related processes such as glycolysis, the TCA cycle, and amino acid metabolism.

• DNA Binding Domain Similarities: The DNA-binding domain of NCU03938, like other zinc-cluster transcription factors in N. crassa, can be expressed as a functional unit. For similar factors, the first 426 bp of the coding sequence effectively binds target DNA sequences .

Understanding these relationships is critical for building a complete model of metabolic regulation in N. crassa and identifying potential applications in metabolic engineering.

What are the common difficulties in achieving functional expression of full-length NCU03938 and how can they be overcome?

Expressing full-length functional NCU03938 presents several challenges that researchers should anticipate:

• Protein Solubility Issues:

  • Challenge: The full 740-residue protein may form inclusion bodies in bacterial expression systems

  • Solution: Optimize expression conditions by lowering temperature (16-20°C), reducing inducer concentration, or using solubility-enhancing tags like MBP or SUMO

  • Alternative: Express functional domains separately, as has been done with the DNA-binding domain (first 426 bp of coding sequence)

• Zinc Coordination:

  • Challenge: Proper folding requires correct coordination of zinc ions

  • Solution: Supplement growth media and purification buffers with ZnCl₂ and maintain reducing conditions with DTT or β-mercaptoethanol to protect cysteine residues

• Codon Usage Differences:

  • Challenge: N. crassa codon bias differs from E. coli

  • Solution: Use specialized strains like Rosetta (DE3) that supply rare tRNAs or optimize the coding sequence for E. coli expression

• Proteolytic Degradation:

  • Challenge: Large multidomain proteins are susceptible to proteolysis

  • Solution: Include protease inhibitors during purification and consider using protease-deficient expression strains

• Functional Verification:

  • Challenge: Confirming that the recombinant protein retains DNA-binding activity

  • Solution: Implement EMSA assays with known or predicted binding sequences to verify functionality post-purification

These approaches have been successfully applied to similar zinc-cluster transcription factors from N. crassa and can be adapted for NCU03938.

How can researchers distinguish between specific and non-specific DNA binding when characterizing NCU03938 targets?

Distinguishing specific from non-specific DNA binding is critical when characterizing NCU03938 targets. The following methodological approaches are recommended:

• Competition Assays in EMSA:

  • Include unlabeled specific oligonucleotides at increasing concentrations

  • Include unlabeled non-specific oligonucleotides at similar concentrations

  • Specific binding should be competitively inhibited only by the specific sequence

• Mutational Analysis:

  • Create systematic mutations in putative binding sites

  • Test binding affinity to these mutated sequences

  • Identify nucleotides critical for recognition

  • This approach has been successful for characterizing binding sites of other N. crassa transcription factors

• DNA Footprinting:

  • Identify regions protected from nuclease digestion

  • Map precise boundaries of protein-DNA interaction

  • Distinguish high-affinity binding sites from low-affinity interactions

• Comparative Genomics:

  • Analyze conservation of binding motifs across related fungal species

  • True functional binding sites tend to be evolutionarily conserved

  • This approach can filter out spurious binding events

• Integration with Expression Data:

  • Correlate binding sites with transcriptional changes

  • Genes with both NCU03938 binding and expression changes represent high-confidence targets

  • This combined approach is powerful as 704 interactions in N. crassa are supported by both binding and expression evidence

By employing these complementary approaches, researchers can build a high-confidence list of specific NCU03938 target genes involved in gluconeogenesis and related metabolic pathways.

What strategies can address the solvent accessibility challenges of NCU03938 in structural studies?

Structural characterization of NCU03938 presents challenges related to solvent accessibility of different protein regions. Based on solvent accessibility studies of other N. crassa proteins, the following strategies are recommended:

• Surface Engineering:

  • Identify residues with high solvent accessibility (similar to ARG27, LYS168, ARG341, ASP115, LYS298, TYR110, LYS49, GLU86, LYS300, and ARG133 in related proteins)

  • Consider mutating surface-exposed hydrophobic residues to hydrophilic ones

  • This can enhance solubility while maintaining core structure and function

• Domain-Based Approach:

  • Focus structural studies on individual domains rather than the full 740-residue protein

  • The DNA-binding domain has been successfully expressed and studied for similar N. crassa transcription factors

• Solvent Condition Optimization:

  • Screen various buffer compositions, pH values, and additives

  • Include stabilizing agents that reduce aggregation

  • Consider the addition of 5-50% glycerol as recommended for storage

• Co-expression with Binding Partners:

  • Express NCU03938 together with interacting proteins or DNA fragments

  • This can stabilize conformations and mask aggregation-prone surfaces

• Accessibility Prediction:

  • Use computational approaches to predict surface accessibility

  • Focus on residues with limited solvent accessibility (similar to VAL72, ILE73, LEU97, GLY178, ALA182, VAL190, ALA191, ASN224, and others identified in related proteins)

  • These residues likely contribute to structural stability and conformational rigidity

By implementing these strategies, researchers can improve the chances of obtaining structural data for NCU03938, which would provide valuable insights into its DNA-binding mechanism and regulatory function.

How can transcriptomic datasets be integrated to identify the complete regulon of NCU03938?

Identifying the complete NCU03938 regulon requires sophisticated integration of multiple data types. Based on successful approaches with other N. crassa transcription factors, the following strategy is recommended:

• Multi-condition RNA-seq Analysis:

  • Compare transcriptomes between wild-type and NCU03938 deletion/overexpression strains

  • Test multiple environmental conditions, particularly varying carbon sources

  • Identify differentially expressed genes across all conditions

  • This approach provides "TF perturbation" evidence that has been valuable in N. crassa network studies

• ChIP-seq Data Integration:

  • Map genome-wide binding sites of NCU03938

  • Correlate binding events with expression changes

  • The 704 interactions in N. crassa supported by both binding and TF perturbation evidence represent high-confidence regulatory relationships

• Network Analysis:

  • Place NCU03938 targets in the context of the broader N. crassa regulatory network

  • Identify targets shared with other transcription factors

  • In N. crassa, network reconstruction has revealed extensive combinatorial regulation of target genes

• Motif Enrichment Analysis:

  • Identify enriched sequence motifs in bound regions

  • Use these motifs to predict additional regulatory targets

  • Validate predictions experimentally using low-throughput methods like EMSA

• Temporal Dynamics:

  • Analyze time-course data to determine direct vs. indirect effects

  • Primary targets show rapid expression changes after TF modulation

  • Secondary targets show delayed responses

This integrated approach leverages both high-throughput and low-throughput methodologies to build a comprehensive understanding of the NCU03938 regulon.

What computational tools are most effective for predicting NCU03938 binding sites in the N. crassa genome?

For effective prediction of NCU03938 binding sites across the N. crassa genome, the following computational approaches and tools are recommended:

• Position Weight Matrix (PWM) Construction:

  • Derive PWMs from experimentally verified binding sites

  • Use tools like MEME Suite to identify overrepresented motifs

  • Refine the matrix using site-directed mutagenesis data

  • This approach was used to characterize binding sites for several N. crassa transcription factors

• Genome-Wide Scanning:

  • Scan the N. crassa genome with the derived PWM

  • Tools like FIMO (Find Individual Motif Occurrences) or HOMER are effective

  • Set appropriate P-value thresholds to balance sensitivity and specificity

• Conservation-Based Filtering:

  • Compare predicted sites across related fungal species

  • Tools like PhyloP or PhastCons can identify evolutionarily conserved regions

  • Conserved sites are more likely to be functionally relevant

• Integrative Genomics Approaches:

  • Combine binding site predictions with:

    • Open chromatin data (ATAC-seq or DNase-seq)

    • Histone modification profiles

    • Transcription start site annotations

  • Sites in accessible chromatin near transcription start sites have higher predictive value

• Machine Learning Models:

  • Train models on known binding sites incorporating sequence and chromatin features

  • Apply to predict genome-wide binding probabilities

  • Validate high-scoring predictions experimentally

The combination of these approaches can significantly improve the accuracy of NCU03938 binding site predictions compared to sequence-based methods alone, as demonstrated by comprehensive network studies in N. crassa .

What are the most promising future research directions for understanding NCU03938 function?

Based on current knowledge and methodological advances, several promising research directions for NCU03938 warrant further investigation:

• Structural Biology Approaches:

  • Determine the three-dimensional structure of NCU03938, particularly its DNA-binding domain

  • Investigate conformational changes upon DNA binding

  • Characterize the zinc coordination chemistry essential for function

• Interactome Mapping:

  • Identify protein-protein interactions of NCU03938

  • Determine if it functions as part of larger regulatory complexes

  • Assess interactions with chromatin remodeling factors

  • Similar studies have revealed complex regulatory interactions for other N. crassa transcription factors

• Post-translational Regulation:

  • Investigate how phosphorylation, acetylation, or other modifications regulate NCU03938 activity

  • Identify signaling pathways that modulate its function in response to metabolic cues

• Comparative Genomics:

  • Extend studies to related filamentous fungi

  • Determine conservation and divergence of NCU03938 function

  • This approach has provided valuable insights into transcriptional regulatory networks across fungal species

• Synthetic Biology Applications:

  • Engineer NCU03938 with altered binding specificity or regulatory output

  • Develop synthetic regulatory circuits incorporating NCU03938 for metabolic engineering

  • Create chimeric transcription factors combining domains from multiple regulators

These research directions would significantly advance our understanding of NCU03938's role in gluconeogenesis regulation and could reveal applications in metabolic engineering and synthetic biology.

How can contradictory experimental results regarding NCU03938 function be reconciled?

When faced with contradictory experimental results concerning NCU03938 function, researchers should employ the following systematic approach to reconciliation:

• Experimental Conditions Analysis:

  • Carefully compare growth conditions, media composition, and carbon sources

  • Minor differences in zinc availability can significantly impact zinc-cluster transcription factor function

  • NCU03938 activity may vary substantially across different metabolic states

• Strain Background Considerations:

  • Genetic background differences can influence transcription factor function

  • Compare results across different N. crassa strains (e.g., ATCC 24698, 74-OR23-1A, CBS 708.71)

  • Secondary mutations may exist in laboratory strains affecting NCU03938 function

• Methodological Variations:

  • Assess differences in:

    • Protein expression systems

    • Purification protocols

    • DNA-binding assay conditions

    • Zinc supplementation during expression and purification

• Data Integration Approach:

  • Weight evidence based on experimental rigor

  • Prioritize results supported by multiple lines of evidence

  • In N. crassa studies, interactions supported by both binding evidence and TF perturbation evidence are considered highest confidence

• Direct Replication Studies:

  • Design experiments specifically to test contradictory findings

  • Include appropriate controls and standardize protocols

  • Consider collaborative cross-laboratory validation studies