Recombinant Neurospora crassa Glutamyl-tRNA (Gln) amidotransferase subunit A, mitochondrial (NCU00660), partial

What is the function of Glutamyl-tRNA (Gln) amidotransferase in Neurospora crassa?

Glutamyl-tRNA (Gln) amidotransferase (AdT) in Neurospora crassa plays a critical role in protein synthesis by catalyzing the conversion of misacylated Glu-tRNA^Gln to correctly charged Gln-tRNA^Gln. This process is part of the indirect aminoacylation pathway, where glutamyl-tRNA synthetase (GluRS) first attaches glutamate to tRNA^Gln, and then the amidotransferase converts this misacylated tRNA to the correctly charged form by adding an amino group derived from glutamine . This two-step pathway is essential in organisms lacking a dedicated glutaminyl-tRNA synthetase (GlnRS) or in compartments like mitochondria that rely on this indirect mechanism for Gln-tRNA^Gln formation.

How is the NCU00660 gene organized within the Neurospora crassa genome?

The NCU00660 gene encoding the Glutamyl-tRNA (Gln) amidotransferase subunit A is part of the extensive Neurospora crassa genome, which consists of approximately 10,082 protein-coding genes distributed across 7 chromosomes . Like many Neurospora genes, NCU00660 likely contains introns, as the genome has an average of 1.7 introns per gene, with an average intron size of 135.4 bp . The gene exists within the context of a genome with 50% G+C content and where approximately 44% of the sequence is protein-coding . The specific chromosomal location and neighboring genes would be important for researchers studying regulatory elements or conducting genome editing experiments.

What is the relationship between NCU00660 and other aminoacyl-tRNA synthetases in Neurospora?

The Glutamyl-tRNA (Gln) amidotransferase functions within a network of aminoacyl-tRNA synthetases that collectively ensure accurate protein translation. While NCU00660 encodes the amidotransferase subunit A, it works in concert with other tRNA synthetases, including the glutamyl-tRNA synthetase (GluRS) . The relationships between these enzymes in Neurospora likely mirror those observed in other organisms, where biochemical experiments have demonstrated the presence of aminoacyl-tRNA synthetases like aspartyl-tRNA synthetase (AspRS) working alongside amidotransferases . This enzymatic network ensures correct aminoacylation of tRNAs, a critical step in maintaining translational fidelity in both cytosolic and mitochondrial protein synthesis.

What are the recommended methods for cloning and expressing recombinant NCU00660?

For cloning and expressing recombinant NCU00660 from Neurospora crassa, researchers should consider a CRISPR/Cas9-based approach for precise gene manipulation. Based on successful systems developed for Neurospora, incorporate the cas9 sequence into the fungal genome for stable expression, and design a specific guide RNA (gRNA) targeting the NCU00660 locus . The gRNA should be an RNA-duplex consisting of crRNA for target sequence recognition and tracrRNA for interaction with Cas9 . When designing your expression system, consider that Neurospora genes have distinct features including an average gene size of 1,673 bp (481 amino acids) and contain multiple introns . For heterologous expression, E. coli systems are commonly used for recombinant amidotransferase production, similar to methods used for isolating amidotransferases from other organisms .

How can CRISPR/Cas9 be used to create specific mutations in NCU00660?

To create specific mutations in NCU00660 using CRISPR/Cas9, implement the user-friendly CRISPR/Cas9 system developed for Neurospora crassa that integrates the cas9 sequence into the fungal genome . Design a guide RNA that specifically targets the NCU00660 region you wish to modify, ensuring it's followed by the protospacer adjacent motif (PAM) 5′-NGG-3′ . For precise edits, provide a repair template containing your desired mutation flanked by homology arms. The system will create double-strand breaks at the target site, which the cell repairs either through non-homologous end joining (creating small insertions or deletions) or homology-directed repair (incorporating your specific mutation) . When designing guide RNAs, be aware that Neurospora's genome has a 50% G+C content, which may influence guide RNA efficiency and specificity .

What purification methods yield the highest activity for recombinant Glutamyl-tRNA (Gln) amidotransferase?

For optimal purification of recombinant Glutamyl-tRNA (Gln) amidotransferase from Neurospora crassa, implement a multi-step chromatography approach. Begin with affinity chromatography using a histidine tag if your recombinant construct includes one. For native protein purification without tags, use ion exchange chromatography with a salt gradient elution, followed by gel filtration to achieve higher purity. When assessing enzymatic activity, prepare an assay system containing 50 mM Hepes⋅KOH (pH 7.5), 25 mM KCl, 15 mM MgCl₂, 5 mM DTT, 1 mM ATP, and 2.5 mM glutamine . This buffer composition has been effectively used for amidotransferase assays from other organisms and can be adapted for the Neurospora enzyme. Measure activity by tracking the conversion of misacylated Glu-tRNA^Gln to correctly charged Gln-tRNA^Gln, quantifying the reaction products using appropriate radioisotope labeling techniques or other sensitive detection methods .

What genomic features surrounding NCU00660 influence its expression and regulation?

The genomic context surrounding NCU00660 likely plays a crucial role in its expression and regulation. Consider analyzing the intergenic regions around the gene, which average 1,953 bp in the Neurospora genome . These regions may contain promoters, enhancers, and other regulatory elements that control NCU00660 expression. When investigating epigenetic regulation, examine the role of histone acetyltransferases (HATs), as Neurospora possesses multiple HATs involved in transcriptional activation and gene silencing, including homologs to TAFII250, Gcn5p, Sas2p, Sas3p, Esa1p, and Elp3p . The presence of these factors suggests sophisticated regulation of gene expression. Additionally, check for tRNA genes near NCU00660, as the Neurospora genome contains 424 tRNA genes, with 57% containing introns . Proximity to tRNA clusters might indicate co-regulation of translation-related genes.

What tRNA species interact with Neurospora crassa Glutamyl-tRNA (Gln) amidotransferase?

Neurospora crassa Glutamyl-tRNA (Gln) amidotransferase specifically interacts with misacylated Glu-tRNA^Gln to convert it to the correctly charged Gln-tRNA^Gln. The Neurospora genome contains 424 tRNA genes capable of decoding all standard amino acids, providing the substrate tRNAs for this process . The enzyme likely recognizes specific structural features of tRNA^Gln that distinguish it from tRNA^Glu, despite both being initially charged with glutamate. For experimental studies, researchers can prepare substrates similar to the methods used for other organisms, where aminoacylated tRNA is isolated by phenol extraction and ethanol precipitation . When analyzing tRNA-protein interactions, consider that approximately 57% of Neurospora tRNA genes contain introns, which may influence the structural features of mature tRNAs and potentially their interactions with the amidotransferase .

How can I establish a reliable activity assay for Neurospora crassa Glutamyl-tRNA (Gln) amidotransferase?

To establish a reliable activity assay for Neurospora crassa Glutamyl-tRNA (Gln) amidotransferase, adapt the established amidotransferase assay protocol used for related organisms. Set up your reaction at 30°C containing 50 mM Hepes⋅KOH (pH 7.5), 25 mM KCl, 15 mM MgCl₂, 5 mM DTT, 1 mM ATP, 2.5 mM glutamine, and your partially purified recombinant enzyme . For substrate preparation, consider using either homologous Neurospora tRNAs or heterologous substrates like [¹⁴C]Glu-tRNA^Gln prepared from Bacillus subtilis, which has been successfully used in similar assays . For optimal results, prepare aminoacylated tRNA through phenol extraction and ethanol precipitation to ensure purity . Quantify the conversion of Glu-tRNA^Gln to Gln-tRNA^Gln using techniques such as thin-layer chromatography or HPLC analysis of amino acids after deacylation. Include appropriate controls, such as reactions without ATP or glutamine, to validate the specificity of your assay.

What are the typical kinetic parameters for Glutamyl-tRNA (Gln) amidotransferase, and how do mutations affect them?

The typical kinetic parameters for Glutamyl-tRNA (Gln) amidotransferase include the Michaelis constant (Km) for its substrates (misacylated Glu-tRNA^Gln, ATP, and glutamine) and the catalytic rate constant (kcat). While specific values for the Neurospora enzyme aren't provided in the search results, researchers should expect Km values in the micromolar range for tRNA substrates and millimolar range for small molecule substrates like ATP and glutamine, based on similar enzymes. Mutations in the NCU00660 gene could significantly alter these parameters depending on their location within the protein structure. Mutations in the active site would likely increase Km values (reduced substrate affinity) or decrease kcat (reduced catalytic efficiency). The exceptionally high mutation rate in Neurospora (3.38 × 10⁻⁶ per bp per generation) means that natural variants may be common . When characterizing mutant enzymes, researchers should perform comprehensive kinetic analyses under varying substrate concentrations and compare multiple parameters to fully understand how specific mutations affect enzyme function.

How does Neurospora crassa Glutamyl-tRNA (Gln) amidotransferase compare to those from other fungi and bacteria?

Neurospora crassa Glutamyl-tRNA (Gln) amidotransferase likely shares structural and functional similarities with homologous enzymes from other organisms while possessing unique features adapted to Neurospora's cellular environment. The search results indicate that similar amidotransferase systems exist in bacteria like Deinococcus radiodurans, which contains both aminoacyl-tRNA synthetases (including AsnRS, GlnRS, and GluRS) and glutamyl-tRNA amidotransferase . When comparing fungal and bacterial amidotransferases, researchers should examine substrate specificities, particularly how these enzymes recognize and process misacylated tRNAs. Heterologous substrates like Bacillus subtilis [¹⁴C]Glu-tRNA^Gln have been successfully used in amidotransferase assays, suggesting some conservation of recognition elements across species . Comparative genomic analyses should consider that approximately 39% of Neurospora protein-coding genes have homologs in Schizosaccharomyces pombe and 37% in Saccharomyces cerevisiae, providing context for evolutionary relationships of this enzyme system across fungal species .

What evolutionary insights can be gained from studying NCU00660 across different species?

Studying NCU00660 across different species offers valuable evolutionary insights into the development and conservation of indirect aminoacylation pathways. The presence of glutamyl-tRNA amidotransferase in diverse organisms from bacteria to fungi suggests an ancient origin of this pathway . Comparative analysis might reveal whether Neurospora's enzyme represents a conserved core mechanism or contains unique adaptations. The Neurospora genome contains approximately 10,082 protein-coding genes, of which only 13% have best bidirectional hits with human proteins, while 22% share such relationships with both S. cerevisiae and S. pombe . This pattern suggests that NCU00660 might show greater conservation among fungi than with distant eukaryotes. Researchers should examine whether the high mutation rate in Neurospora (two orders of magnitude higher than any other non-viral organism) has accelerated the evolution of its aminoacylation systems compared to organisms with lower mutation rates , potentially revealing how essential translation machinery adapts under different evolutionary pressures.

How can I use cross-species complementation to study Glutamyl-tRNA (Gln) amidotransferase function?

To implement cross-species complementation studies for Glutamyl-tRNA (Gln) amidotransferase, design experiments where the Neurospora crassa NCU00660 gene is expressed in amidotransferase-deficient strains of model organisms like Saccharomyces cerevisiae or Escherichia coli. For genetic manipulation in Neurospora, utilize the CRISPR/Cas9 system described in the research, which incorporates the cas9 sequence into the fungal genome and uses a guide RNA for precise targeting . When designing complementation constructs, consider the average gene size in Neurospora (1,673 bp) and the presence of introns (average 1.7 per gene), which may need to be removed for expression in bacterial systems . Evaluate complementation by measuring growth rates and protein synthesis efficiency in the recipient organism. For biochemical confirmation, isolate the recombinant enzyme from complemented strains and assess its activity using the amidotransferase assay conditions described earlier (containing Hepes⋅KOH buffer, KCl, MgCl₂, DTT, ATP, and glutamine) . This approach can reveal functional conservation and species-specific requirements for enzyme activity.

Why might recombinant NCU00660 show low activity in expression systems?

Recombinant NCU00660 might show low activity in expression systems due to several potential factors. First, consider that heterologous expression may not provide the necessary post-translational modifications or proper folding environment for this Neurospora protein. Second, the exceptionally high mutation rate in Neurospora (3.38 × 10⁻⁶ per bp per generation) means that even carefully cloned genes might contain mutations affecting function . Third, if your expression construct includes duplicate sequences, be aware that these could have been affected by RIP (repeat-induced point mutation) if derived from Neurospora genomic DNA, potentially changing critical amino acids . When optimizing expression, adjust buffer conditions to match those used successfully for similar enzymes (50 mM Hepes⋅KOH, pH 7.5, 25 mM KCl, 15 mM MgCl₂) . Also consider that amidotransferase activity requires multiple substrates (misacylated tRNA, ATP, glutamine) and potentially accessory subunits not present in your recombinant system . Enzyme assays should be performed under conditions that closely mimic the physiological environment of Neurospora mitochondria.

How can I resolve contamination issues in purified recombinant NCU00660 preparations?

To resolve contamination issues in purified recombinant NCU00660 preparations, implement a multi-step purification strategy tailored to the specific contaminants observed. If bacterial proteins are the main contaminants, consider adding an ion exchange chromatography step with carefully optimized salt gradients based on the theoretical isoelectric point of NCU00660. For nucleic acid contamination, which can be particularly problematic with tRNA-binding proteins, incorporate a high-salt wash (0.5-1.0 M NaCl) during purification, or treat samples with nucleases followed by an additional gel filtration step. When expressing in fungal systems, be aware that Neurospora produces many enzymes involved in histone modification, such as histone acetyltransferases , which might co-purify with your target protein if they share physical properties. Use activity-based assays to track your protein through purification, measuring the conversion of misacylated tRNA substrates under standardized conditions . For the most challenging purifications, consider alternative tagging strategies or expression systems, keeping in mind that fusion tags may occasionally affect enzyme activity and might need to be removed for final functional studies.

What strategies can overcome the challenges of working with RIP-affected Neurospora strains?

To overcome challenges when working with RIP-affected Neurospora strains, implement a comprehensive strategy addressing the unique features of this genome defense mechanism. First, when designing experiments, be aware that Neurospora has the highest mutation rate of any non-viral organism (3.38 × 10⁻⁶ per bp per generation), with 93-98% of mutations being RIP-associated . To minimize RIP effects, avoid introducing duplicate sequences, as RIP preferentially targets GC-poor long duplicates that interact in three-dimensional space . For genetic modifications of NCU00660, use CRISPR/Cas9 systems specifically developed for Neurospora that allow precise editing without introducing duplicates . When analyzing unexpected results, consider that even non-duplicate sequences may contain mutations in RIP-proficient strains . For critical experiments, use RIP-deficient strains which show over an order of magnitude fewer coding sequence mutations outside duplicated domains . Finally, when cloning NCU00660, verify the sequence of your construct against the reference genome, and be prepared to correct RIP-induced mutations through site-directed mutagenesis to restore wild-type function.

What are promising areas for future research on NCU00660 and related genes?

Promising areas for future research on NCU00660 and related genes include investigating the regulatory networks controlling amidotransferase expression in response to cellular stress and nutrient availability. Explore the potential roles of histone acetyltransferases (HATs) in regulating NCU00660 expression, as Neurospora possesses homologs to various HATs involved in transcriptional activation and gene silencing . Investigate how the exceptionally high mutation rate in Neurospora (3.38 × 10⁻⁶ per bp per generation) affects the evolution of translation-related genes compared to other organisms . Develop comprehensive models of the indirect aminoacylation pathway in Neurospora mitochondria, including the interplay between aminoacyl-tRNA synthetases and amidotransferases. Apply CRISPR/Cas9 technology to create precise mutations in NCU00660 and assess their effects on mitochondrial translation and cellular fitness . Explore potential moonlighting functions of glutamyl-tRNA amidotransferase beyond its canonical role in protein synthesis, as suggested by studies of related enzymes in other organisms.

How might structural biology approaches advance our understanding of NCU00660?

Structural biology approaches would significantly advance our understanding of NCU00660 by revealing the molecular architecture of Neurospora crassa Glutamyl-tRNA (Gln) amidotransferase and its interactions with substrates. X-ray crystallography or cryo-electron microscopy of the purified protein, both alone and in complex with misacylated tRNA substrates, would elucidate the structural basis for substrate recognition and catalysis. These structures could be compared with homologous enzymes from organisms like Deinococcus radiodurans, where biochemical work on glutamyl-tRNA amidotransferase has been reported . Neurospora-specific structural features might explain any unique functional properties of this enzyme. Molecular dynamics simulations based on these structures could provide insights into conformational changes during catalysis. Additionally, hydrogen-deuterium exchange mass spectrometry could map protein-tRNA interaction sites under various conditions. These structural data would provide a foundation for rational design of mutations to test mechanistic hypotheses about enzyme function and could guide the development of specific inhibitors for research purposes.

What applications might emerge from in-depth understanding of tRNA amidotransferases in Neurospora?

In-depth understanding of tRNA amidotransferases in Neurospora could lead to several innovative applications. In biotechnology, engineered amidotransferases might enable incorporation of non-standard amino acids into proteins through manipulated indirect aminoacylation pathways. The knowledge gained could advance synthetic biology efforts to create minimal translation systems by defining essential components of amino acid incorporation machinery. For fungal genetics, deeper understanding of NCU00660 could improve genetic engineering tools for Neurospora, building upon established CRISPR/Cas9 systems . The relationship between amidotransferase activity and Neurospora's exceptionally high mutation rate (3.38 × 10⁻⁶ per bp per generation) might reveal new insights into how translation quality control mechanisms evolve in organisms with different mutation profiles. Additionally, comparative studies between Neurospora amidotransferases and those from pathogenic fungi could potentially identify fungal-specific features of these essential enzymes, possibly providing targets for antifungal development in the longer term.

What are the key takeaways for researchers studying NCU00660?

Researchers studying NCU00660 should recognize several key points that emerge from the available data. First, Glutamyl-tRNA (Gln) amidotransferase represents an essential component of the indirect aminoacylation pathway in Neurospora crassa, functioning within a complex network of enzymes that ensure accurate protein translation. Second, researchers must account for Neurospora's exceptionally high mutation rate (3.38 × 10⁻⁶ per bp per generation) and the potential effects of RIP (repeat-induced point mutation) when designing experiments and interpreting results . Third, the CRISPR/Cas9 system developed specifically for Neurospora provides a powerful tool for precise genetic manipulation of NCU00660 . Fourth, biochemical characterization of the enzyme should follow established protocols for related amidotransferases, including appropriate buffer conditions (Hepes⋅KOH, KCl, MgCl₂, DTT, ATP, and glutamine) . Fifth, comparative analyses with homologous enzymes from other organisms can provide evolutionary insights, considering that approximately 37-39% of Neurospora protein-coding genes have homologs in other model fungi . These considerations form a foundation for rigorous research on this important component of Neurospora's translational machinery.

How does research on NCU00660 contribute to broader understanding of translation mechanisms?

Research on NCU00660 contributes significantly to our broader understanding of translation mechanisms by illuminating the indirect pathway of tRNA aminoacylation, a critical process ensuring translational fidelity. By studying this Neurospora enzyme, researchers gain insights into how organisms maintain accurate protein synthesis when direct charging of tRNA^Gln by a dedicated glutaminyl-tRNA synthetase is unavailable, particularly in organelles like mitochondria. This research connects to fundamental questions about the evolution of the genetic code and translation machinery, especially considering Neurospora's extraordinarily high mutation rate (two orders of magnitude higher than any other non-viral organism) . Understanding how accurate translation persists despite this mutational pressure offers valuable perspectives on the robustness of cellular information processing. Additionally, the amidotransferase represents an example of enzymatic error correction in biological systems, where the misacylated Glu-tRNA^Gln is converted to the correctly charged form through a precise chemical transformation , demonstrating how cells employ multiple layers of quality control to maintain the fidelity of information flow from genes to proteins.