Recombinant Saccharomyces cerevisiae Putative uncharacterized protein YCR097W-A (YCR097W-A)

What is YCR097W-A and where is it located in the S. cerevisiae genome?

YCR097W-A is a putative uncharacterized protein in Saccharomyces cerevisiae. Based on genomic analyses, it is located on chromosome XVI. The gene appears to be positioned near silent mating-type loci, specifically flanking the silent HMR and HML loci along with YCRWDDta12 . This genomic positioning suggests potential involvement in mating-type regulation mechanisms, though its precise function remains to be fully characterized.

What experimental approaches are recommended for initial characterization of YCR097W-A?

For initial characterization of YCR097W-A, a multi-faceted approach is recommended:

• Gene deletion analysis: Generate deletion mutants (ycr097w-aΔ) to observe phenotypic changes. This technique has been successful in characterizing other uncharacterized ORFs in S. cerevisiae .

• Transcriptomic profiling: Perform RNA-seq or microarray analysis comparing wild-type and deletion mutants to identify genes with altered expression. Similar approaches have been productive for characterizing other putative zinc finger proteins .

• Protein localization: Use GFP-tagging to determine subcellular localization, which can provide clues to function.

• Proteomic analysis: Conduct affinity capture-MS experiments to identify potential protein-protein interactions.

• Phenotypic screening: Subject deletion mutants to various stress conditions to observe differential responses compared to wild-type.

How does the genomic context of YCR097W-A inform hypotheses about its function?

The genomic context of YCR097W-A provides important clues about its potential function. YCR097W-A is located near silent mating loci in S. cerevisiae, specifically flanking the HMR and HML loci . This positioning suggests several hypotheses:

• Mating-type regulation: The proximity to silent mating loci suggests YCR097W-A might be involved in silencing mechanisms or mating-type switching.

• Chromatin organization: It may participate in establishing or maintaining chromatin boundaries near these silenced regions.

• Evolutionary significance: The gene's location suggests it might have evolved alongside the mating-type determination system.

• Transcriptional regulation: Similar to other proteins in this genomic region, it might function in regulating gene expression through interaction with silencing factors.

Interestingly, a related observation was made in Candida glabrata, where loci similar to YCR097W-A were found flanking mating-type-like (MTL) genes , suggesting conserved positioning across yeast species.

What approaches can be used to determine if YCR097W-A interacts with the silencing machinery at HM loci?

To investigate potential interactions between YCR097W-A and silencing machinery at HM loci, researchers should consider these methodological approaches:

• Chromatin Immunoprecipitation (ChIP): Using tagged YCR097W-A protein, perform ChIP followed by sequencing (ChIP-seq) to identify genomic binding sites. Compare binding patterns near HMR/HML versus genome-wide.

• Co-immunoprecipitation (Co-IP): Conduct Co-IP experiments to identify direct protein interactions with known silencing factors (Sir2, Sir3, Sir4).

• Genetic interaction screens: Create double mutants of ycr097w-aΔ with deletions of known silencing factors and assess epistatic relationships.

• RNA expression analysis at silent loci: Use RT-qPCR to measure expression of normally silenced genes at HM loci in wild-type versus ycr097w-aΔ strains.

• Synthetic genetic array (SGA) analysis: Perform SGA to identify genome-wide genetic interactions with ycr097w-aΔ, focusing on interactions with genes involved in silencing.

A proposed experimental design would include generating strains with epitope-tagged YCR097W-A, followed by ChIP-seq to identify binding sites near HM loci. Expression of normally silenced genes should be measured in wild-type versus mutant backgrounds using the following RT-qPCR approach:

This approach is similar to methods used in analyzing the mating-type loci in Candida glabrata .

How can transcriptomic analysis help characterize the function of YCR097W-A?

Transcriptomic analysis is a powerful approach to characterize the function of putative uncharacterized proteins like YCR097W-A. Based on successful approaches used for similar proteins , the following methodology is recommended:

• Experimental design: Generate single (ycr097w-aΔ) and double mutants with functionally related genes, along with wild-type controls.

• RNA preparation and analysis: Extract total RNA from exponentially growing cultures under both standard and stress conditions.

• Microarray or RNA-seq analysis: Compare gene expression profiles between wild-type and mutant strains.

• Data analysis: Apply hierarchical clustering analysis (HCA) and functional categorization using Munich Information Center for Protein Sequences (MIPS).

• Validation: Confirm key expression changes using real-time PCR for selected genes.

When a similar approach was applied to characterize other putative zinc finger proteins (YPR013C and YPR015C), researchers found significant alterations in gene expression patterns:

Notably, 80% of alterations in the double mutant were not observed in either single mutant, revealing synergistic effects . This approach could similarly reveal functional pathways influenced by YCR097W-A.

What is the evolutionary significance of YCR097W-A based on comparative genomics?

The evolutionary significance of YCR097W-A can be investigated through comparative genomics approaches. Given limited direct information, we can draw insights from similar analyses:

• Ortholog identification: Search for orthologs across yeast species using BLAST and synteny analysis, particularly focusing on close relatives of S. cerevisiae.

• Sequence conservation analysis: Calculate evolutionary rates (dN/dS ratios) to determine selection pressures.

• Synteny analysis: Examine conservation of gene order surrounding YCR097W-A across species.

• Functional domain prediction: Identify conserved functional domains that might indicate ancestral functions.

An interesting comparative example comes from studies of M dsRNAs in killer yeast systems, where the klus preprotoxin shows high conservation with S. cerevisiae YFR020W ORF, suggesting an evolutionary relationship . Similarly, examining YCR097W-A's conservation pattern could reveal important evolutionary insights.

Researchers should pay particular attention to the conservation of positioning relative to mating-type loci across different yeast species, as similar positioning has been observed between S. cerevisiae and C. glabrata , suggesting functional constraints maintained through evolution.

What are the optimal conditions for expressing recombinant YCR097W-A for structural and functional studies?

For optimal expression of recombinant YCR097W-A, researchers should consider the following methodological approaches:

• Expression system selection:

  • Homologous expression in S. cerevisiae: Use strong inducible promoters (GAL1, CUP1) for native folding and modifications

  • Heterologous expression in E. coli: Consider using BL21(DE3) or Rosetta strains with codon optimization

• Purification strategy:

  • N-terminal or C-terminal tagging (His6, GST, or MBP) based on structural predictions

  • Test multiple tag positions to determine optimal configuration for protein stability

  • Include protease inhibitors during lysis to prevent degradation

• Solubility optimization:

  • If membrane-associated, test various detergents (DDM, CHAPS, Triton X-100)

  • Consider low-temperature induction (16-18°C) to improve folding

  • Test various buffer conditions with different pH values and salt concentrations

• Functional preservation verification:

  • Develop activity assays based on predicted functions

  • Use circular dichroism to confirm proper folding

  • Verify oligomeric state by size exclusion chromatography

For S. cerevisiae expression, researchers should streak cells on fresh YPD agar plates for experimental purposes and use standard growth conditions (30°C, aerobic). If the protein associates with DNA, consider expressing in strains with simplified mating-type loci to avoid confounding interactions.

How can researchers resolve contradictions in YCR097W-A functional data from different experimental approaches?

Resolving contradictions in functional data for YCR097W-A requires systematic analysis and integration of multiple experimental approaches:

Success in resolving such contradictions has been demonstrated in studies of zinc finger proteins where microarray analysis complemented protein interaction data from affinity capture-MS and proteome chip technology, revealing that observed interaction effects manifested in transcriptional regulation patterns .

What bioinformatic pipelines are most effective for predicting the function of YCR097W-A?

For effective functional prediction of YCR097W-A, a multi-layered bioinformatic approach is recommended:

• Sequence-based analysis:

  • PSI-BLAST for distant homology detection

  • InterProScan for motif and domain identification

  • PSIPRED for secondary structure prediction

  • TMHMM for transmembrane region prediction

  • SignalP for signal peptide identification

• Structural prediction:

  • AlphaFold2 for tertiary structure prediction

  • Structure-based function prediction using ProFunc

  • Molecular docking simulations with predicted interactors

• Genomic context analysis:

  • Gene neighborhood conservation across yeast species

  • Co-expression network analysis using publicly available datasets

  • Assessment of synteny and genomic positioning near mating loci

• Integrated analysis:

  • FunCoup for network-based function prediction

  • CAFA-based tools for Gene Ontology term prediction

  • YeastNet for yeast-specific functional inference

The pipeline should particularly focus on the protein's genomic context near silent mating-type loci, as this positioning appears to be conserved between S. cerevisiae and C. glabrata . This conservation suggests functional constraints that might inform prediction algorithms.

A recommended workflow would integrate these approaches in a decision tree format, with higher weight given to experimental evidence from related proteins and genomic context information, which has proven valuable in characterizing other uncharacterized ORFs in yeast .

What emerging technologies might accelerate characterization of YCR097W-A?

Several emerging technologies hold promise for accelerating the characterization of putative uncharacterized proteins like YCR097W-A:

• CRISPR-based technologies:

  • CRISPRi for tunable repression to study dosage effects

  • CRISPRa for overexpression phenotypes

  • CRISPR base editing for studying effects of specific amino acid changes

• Proximity labeling technologies:

  • BioID or TurboID for identifying neighborhood proteins

  • APEX2 for temporal-specific interaction mapping

  • Split-BioID for conditional interaction studies

• Single-cell technologies:

  • scRNA-seq to characterize heterogeneity in response to YCR097W-A deletion

  • scATAC-seq to identify chromatin accessibility changes

• Cryo-EM and integrative structural biology:

  • High-resolution structural determination of YCR097W-A alone and in complexes

  • Integrative modeling combining crosslinking mass spectrometry with computational prediction

• Synthetic biology approaches:

  • Minimal genome studies to determine essentiality in reduced genetic backgrounds

  • Synthetic genetic interaction mapping using enhanced methodology

The most promising approach may be combining CRISPR-based genome editing with high-throughput phenotypic screening, similar to methods that have successfully characterized other zinc finger proteins involved in transcriptional regulation . This would allow for precise genetic manipulation coupled with comprehensive phenotypic analysis.

How should researchers approach publication of findings on YCR097W-A given its uncharacterized status?

When publishing findings on uncharacterized proteins like YCR097W-A, researchers should follow these strategic approaches:

• Evidence integration framework:

  • Present multiple lines of evidence supporting functional claims

  • Clearly distinguish between direct experimental evidence and predictions

  • Use a standardized confidence scoring system for functional assignments

• Comparative contextualization:

  • Position findings within the context of related proteins or systems

  • Discuss evolutionary implications based on comparative genomics

  • Connect to broader biological processes, such as mating-type regulation

• Transparency in limitations:

  • Explicitly acknowledge technical limitations and alternative interpretations

  • Discuss potential confounding factors in experimental approaches

  • Present negative results alongside positive findings

• Data presentation best practices:

• Future direction framing:

  • Propose specific testable hypotheses based on current findings

  • Suggest most critical next experiments for function validation

  • Outline potential broader impacts of complete characterization

Successful examples of this approach include the transcriptomic profiling of zinc finger protein mutants, where researchers clearly distinguished between direct observations and interpretations while suggesting specific follow-up experiments such as ChIP-on-chip assays .

What are the most valuable resources and databases for researching YCR097W-A?

Researchers investigating YCR097W-A should utilize these specialized resources:

• S. cerevisiae-specific databases:

  • Saccharomyces Genome Database (SGD): Comprehensive repository of genetic and molecular biology data

  • YEASTRACT: Transcription factor associations and regulatory networks

  • FungiDB: Integrative genomic database for fungi

  • Yeast Deletion Collection: Access to ycr097w-aΔ strains

• Functional genomics resources:

  • Gene Ontology Resource: Standardized functional annotations

  • STRING database: Protein-protein interaction predictions

  • BioGRID: Curated interaction repository

  • Munich Information Center for Protein Sequences (MIPS): Functional categorization system

• Structural biology tools:

  • PDB: Repository of protein structures

  • AlphaFold DB: AI-predicted structures

  • ModBase: Comparative protein structure models

• Evolutionary analysis resources:

  • Fungal Orthogroups Repository

  • YGOB: Yeast Gene Order Browser for synteny analysis

• Experimental protocol repositories:

  • Protocols.io: Community-contributed yeast protocols

  • AddGene: Plasmids for yeast protein expression