Recombinant Saccharomyces cerevisiae Putative uncharacterized protein YBR292C (YBR292C)

What is YBR292C and where is it located in the S. cerevisiae genome?

YBR292C is a putative protein of unknown function that is conserved across Saccharomyces cerevisiae strains. It is located on Chromosome II spanning positions 784702 to 785073, encompassing 370 base pairs of DNA. The protein is not considered essential for yeast viability based on knockout studies . YBR292C has several synonyms in the literature including YBR2040 and is sometimes simply referred to as "Uncharacterized protein YBR292C" .

How conserved is YBR292C across yeast species?

YBR292C is described as being conserved across S. cerevisiae strains . Conservation across strains suggests functional importance despite being non-essential. When studying YBR292C, researchers should consider analyzing its evolutionary conservation pattern using tools like multiple sequence alignment to identify highly conserved residues that might be crucial for its function. Comparative genomics approaches can reveal the presence of orthologs in other yeast species beyond S. cerevisiae, which could provide insights into its functional role through evolutionary context.

What knockout strains are available for studying YBR292C function?

Several YBR292C knockout strains are commercially available for research purposes. These include:

These knockout strains are maintained in YPD broth with G418 (200 μg/mL) and 15% glycerol . When working with these strains, researchers should note that they are classified as genetically modified micro-organisms (GMMO) under IATA regulations and ship as UN3245, requiring appropriate regulatory compliance.

What expression systems are recommended for recombinant YBR292C production?

For recombinant expression of YBR292C, E. coli-based systems have been successfully employed. The full-length protein (amino acids 1-123) has been expressed with an N-terminal His-tag to facilitate purification . The E. coli expression system offers several advantages for YBR292C production:

• High yield of recombinant protein

• Established purification protocols for His-tagged proteins

• Relatively quick expression timeframe

• Cost-effectiveness compared to yeast-based expression systems

When designing expression constructs, researchers should consider codon optimization for E. coli to improve expression efficiency since the codon usage differs between yeast and bacterial systems.

What are the recommended storage and handling conditions for recombinant YBR292C protein?

Recombinant YBR292C protein requires specific handling and storage conditions to maintain stability and activity:

Before opening, vials should be briefly centrifuged to bring contents to the bottom. After reconstitution, the protein solution should be aliquoted to avoid repeated freeze-thaw cycles, which can degrade protein quality .

What experimental strategies can be employed to determine YBR292C function?

Given that YBR292C remains uncharacterized, several complementary experimental approaches can be used to elucidate its function:

• Phenotypic profiling: Compare growth of wild-type and YBR292C knockout strains under various stress conditions to identify conditions where YBR292C may play a role. This approach has been successful in characterizing other yeast proteins of unknown function.

• Localization studies: Create GFP-tagged versions of YBR292C to determine its subcellular localization, which can provide clues about its function.

• Protein-protein interaction studies: Use techniques such as affinity purification-mass spectrometry (AP-MS), yeast two-hybrid screening, or BioID to identify proteins that physically interact with YBR292C, similar to approaches used for identifying Yih1-binding proteins .

• Transcriptomic analysis: Compare gene expression profiles between wild-type and YBR292C knockout strains to identify differentially expressed genes that might reveal pathways involving YBR292C.

• Evolutionary analysis: Examine the presence and sequence conservation of YBR292C across different yeast species to gain insights into its evolutionary importance.

How can researchers investigate potential regulatory roles of YBR292C?

To investigate whether YBR292C has regulatory functions:

• Chromatin immunoprecipitation (ChIP): If YBR292C potentially binds DNA, ChIP can identify genomic binding sites.

• RNA immunoprecipitation (RIP): To test if YBR292C binds RNA, which could suggest post-transcriptional regulatory functions.

• Genetic interaction mapping: Synthetic genetic array (SGA) analysis can identify genes that interact genetically with YBR292C, revealing functional relationships.

• Phosphoproteomic analysis: Determine if YBR292C is phosphorylated under different conditions, which could indicate involvement in signaling pathways.

• Metabolomic analysis: Compare metabolite profiles between wild-type and knockout strains to identify metabolic pathways affected by YBR292C deletion.

What is known about YBR292C's genomic context and potential co-regulation?

YBR292C is part of the inter-ORF distances in S. cerevisiae that have been analyzed for promoter and terminator sizing. The genomic organization of genes in yeast provides important clues about regulation. YBR292C appears in lists of tandemly orientated ORF pairs separated by specific distances, which can affect their regulation .

When studying tandemly arranged genes, researchers should be aware that genes separated by short intergenic regions may experience transcriptional interference (TI), where transcription of the upstream gene can affect the downstream gene expression. Experimental validation through RT-PCR using primers spanning consecutive ORFs can help determine if polycistronic transcripts exist .

What bioinformatic approaches can predict potential functions of YBR292C?

Several computational approaches can provide functional insights for uncharacterized proteins like YBR292C:

• Sequence-based predictions: Tools like InterProScan, PFAM, and SMART can identify conserved domains, motifs, or signatures in the protein sequence.

• Structure prediction: AlphaFold2 or I-TASSER can generate three-dimensional structural models that may reveal structural similarities to proteins of known function.

• Transmembrane topology prediction: Tools like TMHMM or Phobius can predict membrane-spanning regions, which appears relevant given YBR292C's sequence characteristics.

• Gene context analysis: Examining neighboring genes and their functions can provide contextual clues, especially relevant given YBR292C's appearance in studies of tandemly arranged ORFs .

• Gene co-expression networks: Analyzing which genes show similar expression patterns to YBR292C can suggest functional associations.

• Characteristic sequence analysis: Methods similar to those used for protein coding gene finding in yeast genomes can provide insights into functional elements within the sequence .

How can mass spectrometry be optimized for studying YBR292C interactions?

When designing mass spectrometry experiments to study YBR292C interactions:

• Bait protein preparation: Express YBR292C with appropriate tags (e.g., His-tag) that enable efficient purification while minimizing interference with native interactions .

• Crosslinking approaches: Consider using chemical crosslinkers to capture transient interactions before cell lysis.

• Control experiments: Include appropriate negative controls such as purifications from strains expressing only the tag or an unrelated tagged protein.

• Quantitative approaches: Implement SILAC or TMT labeling to distinguish true interactors from background contaminants.

• Data analysis: Apply stringent statistical filters and compare against databases of common mass spectrometry contaminants.

• Validation studies: Confirm key interactions using orthogonal methods such as co-immunoprecipitation or proximity ligation assays.

How should researchers interpret potential functional data for YBR292C in the context of it being non-essential?

When interpreting functional data for YBR292C:

• Functional redundancy: Consider the possibility that YBR292C function may be compensated by other genes in knockout strains, explaining its non-essential nature .

• Condition-specific functions: Although YBR292C is non-essential under standard laboratory conditions, it may be important under specific environmental, stress, or developmental conditions not typically tested.

• Subtle phenotypes: Look for quantitative rather than qualitative phenotypes, such as slight growth disadvantages or stress sensitivity that might not be obvious in standard assays.

• Systems-level effects: Consider examining effects on multiple cellular processes simultaneously, as the impact of YBR292C might be distributed across several pathways.

• Evolutionary context: Evaluate the conservation pattern of YBR292C across different yeast species and strains to understand its evolutionary significance despite being non-essential.

What are common technical challenges when working with uncharacterized proteins like YBR292C?

Researchers working with uncharacterized proteins like YBR292C often encounter these technical challenges:

• Protein expression difficulties: Uncharacterized proteins may express poorly or form inclusion bodies in heterologous systems. Optimization strategies include trying different expression strains, induction conditions, or fusion tags.

• Protein solubility issues: If YBR292C contains hydrophobic regions, it may have limited solubility. Consider using detergents or amphipathic additives to improve solubility.

• Antibody generation: Without commercial antibodies, researchers need to generate custom antibodies, which can be challenging without knowledge of immunogenic epitopes.

• Functional assays: Without knowing the function, designing appropriate activity assays can be difficult. Consider broad screening approaches or activity-based protein profiling methods.

• Data interpretation: Results may be challenging to interpret without a framework of expected outcomes. Careful experimental design with positive and negative controls is essential.

How does YBR292C research relate to studies of other uncharacterized yeast proteins?

Approximately 1000 of the ~6000 yeast genes remain uncharacterized or poorly characterized despite the extensive study of S. cerevisiae as a model organism. Approaches for studying YBR292C can be informed by successful strategies for other uncharacterized yeast proteins:

• Systematic approaches: Large-scale studies have identified protein complexes, genetic interactions, and localization patterns for many uncharacterized yeast proteins. YBR292C should be examined in the context of these datasets.

• Comparative genomics: Methods that have successfully characterized other yeast proteins through cross-species comparison can be applied to YBR292C.

• Building on partial information: Even limited information about YBR292C, such as its appearance in lists of tandemly oriented ORFs , can provide starting points for more directed studies.

• Genome-scale methods: Techniques like finding protein coding genes based on characteristic sequences can be complemented with experimental validation.

What controls and validation steps are critical when making functional claims about YBR292C?

When making functional claims about YBR292C, researchers should implement these controls and validation steps:

• Complementation studies: Confirm that phenotypes observed in YBR292C knockout strains can be rescued by reintroducing the wild-type gene.

• Multiple knockout strains: Verify phenotypes in knockout strains with different genetic backgrounds to ensure observations are not strain-specific.

• Point mutations: Create point mutations in conserved residues to identify functionally important amino acids.

• Orthogonal methods: Confirm protein interactions or localization using multiple independent techniques.

• Statistical robustness: Ensure adequate biological and technical replicates with appropriate statistical analysis.

• Publication of negative results: Document approaches that did not yield results to guide future research and avoid duplication of effort.