Recombinant Probable rRNA maturation factor (WS0555)

What is Recombinant Probable rRNA maturation factor (WS0555) and what is its role in ribosome biogenesis?

Recombinant Probable rRNA maturation factor (WS0555), also known by its target name ybeY, is a protein derived from Wolinella succinogenes (strain ATCC 29543 / DSM 1740 / LMG 7466 / NCTC 11488 / FDC 602W). WS0555 plays a crucial role in ribosomal RNA processing and maturation, participating in the complex assembly pathway of functional ribosomes .

The protein functions as a maturation factor that facilitates the proper processing of rRNA precursors, particularly in the formation of the 5S RNP (ribonucleoprotein) complex. Similar to other rRNA maturation factors, WS0555 is involved in the critical anchoring steps that occur during the transition of pre-ribosomes from the nucleolus to the nucleoplasm, enabling the proper folding of rRNA helices and subsequent assembly of ribosomal subunits .

Methodologically, researchers can confirm WS0555's function through complementation assays in knockout strains, where reintroduction of the functional protein should restore proper rRNA processing patterns that can be visualized using Northern blot analysis or RNA sequencing approaches.

What expression systems are recommended for optimal production of WS0555?

Several expression systems can be utilized for WS0555 production, each with distinct advantages depending on your experimental requirements:

E. coli remains the most common system for WS0555 expression, as evidenced in the product specifications . For optimal results, BL21(DE3) strain is frequently employed, though Rosetta-GAMI strains may improve expression of proteins with rare codons .

Standard methodology involves transformation of the expression vector into the chosen host, followed by induction with IPTG (for E. coli), cell harvesting, lysis, and purification via affinity chromatography using the appropriate tag system (His, FLAG, GST, etc.) as per the fusion expression design .

How should WS0555 samples be handled to ensure stability and activity?

Proper handling of WS0555 is critical for maintaining its structural integrity and functional activity. Based on the product information, the following methodological approaches are recommended:

For reconstitution:

• Centrifuge the vial briefly before opening to bring contents to the bottom

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

• Add glycerol to a final concentration of 5-50% (50% being the default recommendation)

• Aliquot for long-term storage to avoid repeated freeze-thaw cycles

Storage conditions:

• Lyophilized form: Stable for 12 months at -20°C/-80°C

• Liquid form: Stable for 6 months at -20°C/-80°C

• Working aliquots: Can be stored at 4°C for up to one week

Activity assessment methods should include functional assays such as in vitro rRNA processing assays or ribonuclease protection experiments to confirm that the protein retains its maturation factor capabilities after storage and handling.

What methods are recommended for assessing the purity and identity of recombinant WS0555?

To ensure the quality of WS0555 preparations, researchers should employ multiple complementary analytical techniques:

For SDS-PAGE analysis, a 12-15% gel is typically recommended for optimal resolution of proteins in this molecular weight range. For Western blotting, antibodies against the fusion tag (His, FLAG, etc.) can be used if specific antibodies against WS0555 are unavailable .

Identity confirmation can be further strengthened through proteomic approaches such as peptide mass fingerprinting or LC-MS/MS sequencing to verify the amino acid sequence against the reference database entry (UniProt Q7MSD7) .

How can WS0555 be used as a tool to study ribosome biogenesis mechanisms?

WS0555 serves as an excellent model for investigating ribosome assembly pathways, particularly the critical steps involving 5S RNP integration. To leverage WS0555 in such studies, several methodological approaches can be employed:

• Proximity-dependent labeling: Using BioID or APEX2 fusions with WS0555 to identify proximal proteins during ribosome assembly, providing a spatiotemporal map of the process.

• Cryo-electron microscopy: Capturing structural snapshots of WS0555-associated pre-ribosomal complexes at different maturation stages to visualize conformational changes.

• Pulse-chase experiments: Using radioisotope or stable isotope labeling to track the kinetics of WS0555 association with nascent rRNA transcripts.

• Conditional depletion systems: Implementing auxin-inducible or tetracycline-regulated systems to control WS0555 expression and monitor consequent effects on ribosome assembly intermediates.

Research indicates that WS0555 and similar maturation factors are involved in critical transitions, including the rotation of the 5S RNP and maturation of functional centers such as the peptidyl transferase center (PTC) and the nascent polypeptide exit tunnel (NPET) . By manipulating WS0555 levels or activity, researchers can dissect the temporal sequence of these events and identify regulatory checkpoints in ribosome biogenesis.

A methodological workflow might include:

• Generate cell lines with tagged or conditionally expressed WS0555

• Induce depletion or expression at defined timepoints

• Isolate pre-ribosomal particles using sucrose gradient centrifugation

• Analyze rRNA processing by Northern blotting and next-generation sequencing

• Identify associated proteins through mass spectrometry

• Visualize structural conformations via cryo-EM

What experimental approaches can determine the RNA binding specificity of WS0555?

Understanding the RNA-binding properties of WS0555 is crucial for elucidating its precise role in rRNA maturation. Several complementary methodologies can characterize this specificity:

Research on similar RNA maturation factors suggests that the RNA recognition motif (RRM) of WS0555 likely binds to specific rRNA regions, potentially in the 5S rRNA or nearby structures . CLIP-seq analysis would be particularly valuable for mapping these interactions in cellular contexts, while in vitro approaches like EMSA and filter binding can quantify binding parameters under controlled conditions.

For a comprehensive characterization, researchers should:

• Generate recombinant WS0555 with and without mutations in predicted RNA-binding residues

• Prepare labeled rRNA fragments representing different domains of the ribosomal RNA

• Perform binding assays under varying ionic conditions

• Use competition assays to determine specificity

• Validate binding sites through mutational analysis of both protein and RNA

How do post-translational modifications impact WS0555 function in rRNA processing?

Post-translational modifications (PTMs) can significantly alter protein function, and understanding their impact on WS0555 requires systematic investigation through the following methodological approaches:

• Identification of PTMs: Employ mass spectrometry-based proteomics (LC-MS/MS) to identify and map PTMs on WS0555 isolated from different cellular contexts. Phosphorylation, methylation, acetylation, and ubiquitination should be specifically monitored.

• Site-directed mutagenesis: Generate non-modifiable mutants by replacing modified residues (e.g., Ser/Thr to Ala for phosphorylation sites) and assess functional consequences.

• Phosphomimetic mutations: Create constitutively "modified" versions (e.g., Ser/Thr to Asp/Glu for phosphorylation) to study the effects of persistent modification.

• In vitro modification: Use purified kinases, acetyltransferases, or other modifying enzymes to generate defined modifications in recombinant WS0555.

• Modification-specific antibodies: Develop or utilize antibodies that specifically recognize modified forms of WS0555 for monitoring modification status in different conditions.

While specific PTM data for WS0555 is not directly presented in the search results, research on related rRNA maturation factors suggests that phosphorylation can regulate subcellular localization and timing of action during ribosome biogenesis. For instance, cell cycle-dependent phosphorylation of nucleolar proteins like NIFK affects their participation in rRNA processing .

A comprehensive experimental design would include:

• Mapping all PTMs present on WS0555 under normal and stress conditions

• Correlating modification patterns with cell cycle stages and growth conditions

• Assessing how modifications affect WS0555 localization, RNA binding affinity, and protein-protein interactions

• Determining the enzymes responsible for each modification

• Testing how modification status correlates with rRNA processing efficiency

What are the methodological approaches for studying WS0555 involvement in quality control of ribosome assembly?

WS0555 may participate in quality control mechanisms that ensure only properly assembled ribosomes progress to maturation. To investigate this aspect, researchers can implement these methodological strategies:

• Ribosome profiling: Apply ribosome profiling techniques to WS0555-depleted cells to assess global translation effects and identify specific mRNAs affected by ribosome quality defects.

• Polysome analysis: Perform sucrose gradient separation of polysomes from cells with normal or depleted WS0555 to evaluate ribosome assembly states and translation activity.

• Nucleolar stress monitoring: Assess p53 activation, RPL5/RPL11 release, and other nucleolar stress markers in response to WS0555 disruption, as these pathways often signal ribosome assembly defects.

• Pulse-chase labeling: Track newly synthesized rRNA processing and maturation using metabolic labeling with 5-fluorouracil or 32P to identify specific steps affected by WS0555 dysfunction.

• Cryo-EM structural analysis: Compare structures of pre-ribosomes isolated from control and WS0555-depleted conditions to identify conformational defects.

Research indicates that disruption of rRNA maturation factors can lead to p53-dependent G1 arrest through the RPL5/RPL11-mediated nucleolar stress pathway, suggesting their importance in quality control mechanisms . WS0555 may similarly participate in monitoring the fidelity of specific ribosome assembly steps.

A systematic investigation would include:

• Generating conditional WS0555 knockout or knockdown systems

• Monitoring cell cycle progression using flow cytometry

• Analyzing pre-rRNA processing patterns by Northern blotting

• Assessing p53 pathway activation through Western blotting and reporter assays

• Characterizing structural abnormalities in pre-ribosomes using cryo-EM

• Measuring the half-life of defective pre-ribosomes to assess degradation kinetics

How can computational approaches enhance our understanding of WS0555 function and evolution?

Computational methodologies offer powerful tools for investigating WS0555 function and evolutionary history:

• Homology modeling and molecular dynamics: Generate structural models of WS0555 based on homologous proteins and simulate its interactions with rRNA to predict binding modes and conformational changes.

• Phylogenetic analysis: Trace the evolutionary history of WS0555 across diverse bacterial lineages to identify conserved domains and lineage-specific adaptations.

• Machine learning for functional prediction: Apply statistical relational AI approaches to predict WS0555 interactions and functions based on patterns extracted from biomedical literature and experimental data.

• Network analysis: Construct protein-protein and protein-RNA interaction networks to position WS0555 within the broader context of ribosome assembly machinery.

• Coevolution analysis: Identify correlated evolutionary changes between WS0555 and its interaction partners (both proteins and rRNA) to map functional interfaces.

Statistical relational AI approaches, as described in the OntoUSP system, can extract knowledge from biomedical abstracts relating to WS0555 and similar proteins, enabling the discovery of previously unrecognized functional connections . These methods can build hierarchical knowledge structures that reveal how WS0555 relates to broader categories of maturation factors.

A comprehensive computational workflow might include:

• Constructing multiple sequence alignments of WS0555 homologs

• Building phylogenetic trees to trace evolutionary history

• Identifying conserved motifs and predicting structure using AlphaFold or similar tools

• Docking models with rRNA substrates

• Simulating dynamics of the complex

• Applying text mining to extract relationships from literature

• Integrating diverse data types to generate testable hypotheses about function