Recombinant Saccharomyces cerevisiae Putative uncharacterized protein YNR005C (YNR005C)

What is known about the basic structure of YNR005C protein?

YNR005C is a putative uncharacterized protein in Saccharomyces cerevisiae with a full length of 134 amino acids. It is currently classified as a questionable open reading frame (ORF) with unknown function . The protein can be produced recombinantly in E. coli with a His-tag, which facilitates purification and subsequent structural studies . When approaching uncharacterized proteins like YNR005C, initial characterization typically involves sequence analysis to identify conserved domains, secondary structure prediction, and examination of physicochemical properties.

How can researchers obtain recombinant YNR005C for experimental studies?

Recombinant YNR005C can be produced using E. coli expression systems with appropriate tags for purification. The His-tagged full-length protein (1-134 amino acids) has been successfully expressed in E. coli . For researchers interested in producing their own recombinant protein, the following methodological approach is recommended:

• Clone the YNR005C gene into an appropriate expression vector with a tag system (His-tag being commonly used)

• Transform into an E. coli expression strain

• Induce expression under optimized conditions

• Purify using affinity chromatography (Ni-NTA columns for His-tagged proteins)

• Perform quality control via SDS-PAGE and mass spectrometry to confirm identity and purity

What computational approaches can predict potential functions of YNR005C?

For uncharacterized proteins like YNR005C, computational prediction serves as a critical first step. Researchers should employ:

• Sequence homology analysis: Compare with characterized proteins to identify potential functional domains

• Structural prediction: Use tools like AlphaFold or Rosetta to model the three-dimensional structure

• Phylogenetic analysis: Examine evolutionary relationships with proteins of known function

• Gene neighborhood analysis: Examine genomic context for functional clues

• Protein-protein interaction prediction: Identify potential binding partners

It's worth noting that YNR005C has sequence similarity to S. cerevisiae sorbitol dehydrogenase and to xylitol dehydrogenase of Pichia stipitis, which provides a starting point for functional hypothesis development .

How can transcriptomic analysis help elucidate the function of YNR005C?

Two-dimensional transcriptome analysis in chemostat cultures represents a powerful approach to understand YNR005C expression patterns. Based on methodologies described in the literature, researchers should:

• Establish chemostat cultures under varied nutrient limitations (carbon, nitrogen, sulfur, and phosphorus) and oxygen conditions (aerobic and anaerobic)

• Extract RNA and perform microarray or RNA-seq analysis

• Analyze differential expression patterns using statistical methods that account for false discovery rates

• Look for co-expression with genes of known function

• Identify regulatory motifs in the promoter region of YNR005C

This approach allowed researchers to identify 3,169 genes (52% of the yeast genome) that showed significant differential expression across various conditions, providing a framework for understanding gene function in context .

What CRISPR-Cas9 strategies can be used to study YNR005C function?

CRISPR-Cas9 technology provides versatile tools for studying uncharacterized genes like YNR005C. Researchers can:

• Generate knockout strains to observe phenotypic effects using the EasyClone-MarkerFree vector toolkit

• Create precise point mutations to examine specific amino acid contributions to function

• Introduce fluorescent tags for localization studies

• Implement conditional expression systems

The EasyClone-MarkerFree vector toolkit allows marker-less integration of genes into S. cerevisiae via CRISPR-Cas9 with 90-100% targeting efficiency for single gene insertions and 60-70% for multiple genes . A typical protocol would involve:

• Design of guide RNA targeting YNR005C locus

• Construction of repair templates containing desired modifications

• Co-transformation of Cas9 plasmid (pCfB2312), gRNA helper vector, and repair template

• Selection on YPD agar containing G418 (200 mg/L) and nourseothricin (100 mg/L)

• Verification by colony PCR

How can protein-protein interaction studies help characterize YNR005C?

Understanding the interaction partners of YNR005C can provide crucial insights into its function. Recommended methodological approaches include:

• Yeast two-hybrid screening to identify binary interactions

• Affinity purification coupled with mass spectrometry (AP-MS) to identify protein complexes

• Proximity-dependent biotin labeling (BioID) to identify proteins in the same subcellular neighborhood

• Bimolecular fluorescence complementation to validate specific interactions in vivo

These interactions can be detected through various methods including yeast two-hybrid, co-immunoprecipitation, and pull-down assays , with subsequent analysis to build an interaction network that may suggest functional roles.

Could YNR005C function as a dehydrogenase based on sequence homology?

Given the sequence similarity between YNR005C and sorbitol/xylitol dehydrogenases, an enzymatic function hypothesis warrants investigation. YLR070c, another previously uncharacterized ORF in S. cerevisiae, showed high sequence similarity to sorbitol dehydrogenase and xylitol dehydrogenase, and overexpression resulted in confirmed xylitol dehydrogenase activity .

To test if YNR005C has similar dehydrogenase activity, researchers should:

• Express and purify recombinant YNR005C

• Perform enzyme assays with various substrates including polyols (sorbitol, xylitol)

• Test cofactor preferences (NAD+/NADH vs. NADP+/NADPH)

• Determine kinetic parameters if activity is detected

• Compare with known dehydrogenases like YLR070c, which has the following parameters:

  • D-xylulose Km = 1.1 mM

  • NADH Km = 240 μM (at pH 7.0)

  • Xylitol Km = 25 mM

  • NAD Km = 100 μM (at pH 9.0)

How does oxygen availability affect YNR005C expression and function?

Oxygen-responsive gene regulation is a significant aspect of yeast metabolism. To investigate how YNR005C responds to oxygen availability:

• Culture S. cerevisiae under both aerobic and anaerobic conditions

• Monitor YNR005C expression at transcriptional and protein levels

• Analyze promoter regions for known oxygen-responsive elements such as:

  • Upc2p binding site (CGTTT)

  • Rox1p binding site (ATTGTTC)

  • Novel motifs like AAGGCAC

• Perform chromatin immunoprecipitation (ChIP) to identify transcription factors binding to the YNR005C promoter

• Create reporter constructs to quantify promoter activity under various oxygen conditions

What is the impact of YNR005C knockout on cellular metabolic networks?

Understanding the system-wide effects of YNR005C deletion requires comprehensive metabolic analysis:

• Generate YNR005C deletion strains using CRISPR-Cas9 technology

• Perform growth phenotyping under various carbon sources and stress conditions

• Conduct transcriptome analysis of wild-type versus deletion strains

• Implement metabolomics approaches to identify altered metabolite pools

• Use 13C metabolic flux analysis to detect changes in carbon flow through central metabolism

Similar approaches have been used to characterize other uncharacterized proteins in bacteria, where depletion of certain membrane proteins led to loss of viability, suggesting essential functions in maintaining cell envelope integrity .

How can YNR005C studies inform synthetic biology applications in S. cerevisiae?

S. cerevisiae is an established industrial host for recombinant protein production, fuels, and chemicals. Understanding YNR005C could contribute to these applications through:

• Assessment of YNR005C as a potential metabolic engineering target

• Integration of YNR005C manipulation with established pathways

• Evaluation of YNR005C-centered interventions on production metrics

For example, when engineering yeast for 3-hydroxypropionic acid production, researchers tested different acetyl-CoA supply strategies requiring overexpression of three to six genes . Similar approaches could incorporate YNR005C if it proves relevant to target pathways.

What high-throughput screening methods can identify conditions where YNR005C is essential?

Determining conditions under which YNR005C becomes essential could reveal its function. Methodological approaches include:

• Chemical genomics screening with YNR005C deletion strains across diverse compounds

• Environmental stress screening (temperature, pH, osmotic pressure)

• Synthetic genetic array (SGA) analysis to identify genetic interactions

• Competitive growth assays in mixed populations under various conditions

• Barcode-based pooled screens with condition-specific selection

How can integrative multi-omics approaches enhance understanding of YNR005C?

A comprehensive understanding of YNR005C requires integration of multiple data types:

• Combine transcriptomics, proteomics, and metabolomics data

• Implement network analysis to position YNR005C within cellular systems

• Apply machine learning to predict function from integrated datasets

• Use temporal analyses to capture dynamic behaviors

• Correlate phenotypic outcomes with molecular signatures

The iTRAQ (isotope tagging for relative and absolute quantification) approach coupled with multidimensional liquid chromatography and tandem mass spectrometry (2D-LC/MS/MS) has been successful in determining uniformly and differentially expressed proteins , and could be applied to studies involving YNR005C.

What are the most promising approaches for definitively characterizing YNR005C function?

Based on current knowledge, the most promising research strategies include:

• Detailed sequence analysis and comparison with xylitol/sorbitol dehydrogenases

• Systematic biochemical assays to test dehydrogenase activity with various substrates

• High-resolution structural determination using X-ray crystallography or cryo-EM

• In vivo localization and dynamic studies using fluorescently tagged variants

• Integration of genetic interaction data with biochemical findings

How can researchers contribute YNR005C findings to the scientific community?

To maximize the impact of YNR005C research:

• Submit structural data to the Protein Data Bank

• Contribute functional annotations to the Saccharomyces Genome Database

• Share reagents through repositories like Addgene

• Use standardized experimental protocols to ensure reproducibility

• Implement FAIR (Findable, Accessible, Interoperable, Reusable) data principles