Recombinant Mannheimia succiniciproducens 30S ribosomal protein S13 (rpsM)

What is the basic structure and function of 30S ribosomal protein S13 in bacterial systems?

30S ribosomal protein S13 is a critical component of the small ribosomal subunit in bacteria. The protein consists of approximately 122 amino acids, as evidenced by the full-length sequence data available for homologous proteins . In structural studies, S13 has been positioned in the head of the 30S subunit, more than 100 Å away from proteins like S20 which reside near the bottom of the body of the 30S subunit .

Functionally, S13 contributes to:

• Stabilization of the tertiary structure of 16S rRNA

• Facilitation of proper ribosome assembly

• Maintenance of translational fidelity

• Potential involvement in interactions between the 30S and 50S subunits

S13 belongs to the 3' major domain family of proteins and is part of the S7 assembly branch in the ribosomal assembly pathway, with biochemical studies demonstrating that S13 can bind to 16S rRNA in the presence of S7 .

What expression systems are recommended for recombinant production of M. succiniciproducens S13 protein?

Based on established protocols for ribosomal proteins, several expression systems can be considered:

Yeast expression systems have been successfully used for the production of recombinant ribosomal proteins similar to S13, as indicated in the available datasheet for a homologous protein . For M. succiniciproducens S13, specific optimization of codon usage, induction conditions, and purification protocols may be necessary based on the protein's characteristics.

What purification strategies yield the highest quality recombinant S13 protein?

For optimal purification of recombinant S13 protein, researchers should consider:

• Initial clarification: Centrifugation at high speeds after cell lysis

• Affinity chromatography: His-tag or other fusion tags depending on the expression construct

• Ion exchange chromatography: To remove nucleic acid contamination

• Size exclusion chromatography: For final polishing

The expected purity should exceed 85% as measured by SDS-PAGE, comparable to similar ribosomal proteins . The purified protein should be stored with 5-50% glycerol at -20°C/-80°C, with liquid formulations having a typical shelf life of approximately 6 months, and lyophilized forms extending to 12 months .

How can researchers verify the functional activity of purified recombinant S13 protein?

Functional validation requires multiple approaches:

• RNA binding assays: Using base-specific chemical footprinting and primer extension analysis to confirm binding to 16S rRNA, similar to techniques used in previous S13 studies .

• In vitro reconstitution experiments: Demonstrating incorporation into partially assembled 30S subunits.

• Assembly dependency tests: Verifying S13's position in the assembly hierarchy by examining its ability to bind in the presence or absence of other ribosomal proteins, particularly S7 .

• Structural integrity verification: Using circular dichroism or thermal shift assays to confirm proper folding.

What is the role of S13 in the assembly pathway of the 30S ribosomal subunit?

Contrary to earlier assembly maps that positioned S13 as dependent on S20, more recent research demonstrates that S13 is actually part of the S7 assembly branch . This positions S13 more logically in accordance with its physical location in the 30S head.

The assembly pathway proceeds as follows:

• Initial binding of primary binding proteins, including S7

• S7 creates a nucleation site for subsequent protein binding in the head domain

• S13 associates with the forming ribonucleoprotein complex, dependent on prior S7 association

• S13 further interacts with other members of the S7 assembly branch

This places S13 in a new location in the 30S subunit assembly map that is more consistent with structural and biochemical data .

How does heat stress affect S13 incorporation during ribosome biogenesis?

Heat stress significantly impacts the late stages of 30S ribosomal subunit biogenesis. Research has shown that exposing bacterial cells to elevated temperatures (45°C) leads to the accumulation of 21S ribosomal particles, which are precursors to mature 30S subunits .

During heat shock:

• The availability of DnaK chaperone proteins devoted to ribosome assembly becomes limited

• This limitation affects the incorporation of late assembly proteins, potentially including S13

• The 21S particles accumulate as assembly intermediates

These heat-stress affected precursors provide valuable research tools for studying authentic assembly intermediates and the role of specific proteins like S13 in the maturation process .

What is the relationship between molecular chaperones and S13 during ribosome assembly?

The DnaK chaperone system plays a crucial role in facilitating 30S ribosomal subunit assembly, particularly under stress conditions. Research indicates:

• DnaK assists in the incorporation of S13 and other late assembly proteins into the forming 30S subunit

• In the absence of functional DnaK chaperones, authentic precursors to ribosomal subunits accumulate

• Heat stress reduces the availability of DnaK for ribosome assembly, affecting the integration of proteins like S13

Understanding this relationship is essential for researchers working with recombinant S13, as proper folding and incorporation may require chaperone assistance for in vitro reconstitution experiments.

How can recombinant S13 be utilized in metabolic engineering studies of M. succiniciproducens?

M. succiniciproducens has significant industrial importance for the production of succinic acid and other metabolites . While direct evidence linking S13 to metabolic engineering is limited in the search results, researchers could explore:

These approaches could complement existing metabolic engineering strategies such as elementary mode analysis (EMC) that have been used to identify gene targets like zwf for improving succinic acid production in M. succiniciproducens .

What methodologies are most effective for studying S13 interactions with other ribosomal components?

To investigate S13's interactions with other ribosomal components, researchers should consider:

• Cryo-electron microscopy (cryo-EM): This technique has been successfully employed to determine the structures of ribosomal particles and precursors, revealing protein contents and arrangements .

• Quantitative proteomics: Mass spectrometry-based proteomics allows for relative quantification of proteins in ribosomal complexes, identifying binding partners and assembly dependencies .

• Chemical crosslinking coupled with mass spectrometry: This approach can capture transient interactions between S13 and other ribosomal proteins or rRNA.

• In vitro reconstitution with labeled components: Using fluorescently or isotopically labeled S13 and potential binding partners to track assembly in real-time.

How can researchers engineer S13 mutants to investigate translation efficiency in M. succiniciproducens?

Creating and studying S13 mutants requires a systematic approach:

• Site-directed mutagenesis: Target conserved residues identified through sequence alignment with well-studied homologs.

• In vivo complementation assays: Use S13-depleted strains complemented with mutant variants to assess functional impacts.

• Ribosome profiling: Measure translation efficiency across the transcriptome in strains with wild-type versus mutant S13.

• In vitro translation systems: Reconstitute ribosomes with mutant S13 proteins to directly assess effects on translation rate and accuracy.

These approaches could provide valuable insights into how S13 contributes to translation efficiency, particularly for mRNAs encoding enzymes involved in the production of industrially important metabolites like succinic acid and malic acid in M. succiniciproducens .

What are the major technical challenges in working with recombinant M. succiniciproducens S13?

Researchers working with recombinant S13 should be aware of several challenges:

• Protein stability: Ribosomal proteins often have limited stability when isolated from their natural RNA environment. Storage recommendations include using 5-50% glycerol and avoiding repeated freeze-thaw cycles .

• RNA contamination: Purification must effectively remove bound nucleic acids that can co-purify with ribosomal proteins.

• Functional validation: Confirming that recombinant S13 retains native functionality requires sophisticated assays that may not be widely available.

• Species-specific interactions: Knowledge from model organisms like E. coli may not directly translate to M. succiniciproducens S13 due to species-specific adaptations.

How can structural studies of S13 contribute to understanding M. succiniciproducens metabolism?

While the connection between ribosomal proteins and metabolism is indirect, structural studies of S13 could provide insights by:

• Revealing how translation efficiency of key metabolic enzymes might be regulated

• Identifying potential sites for engineering to optimize expression of pathways relevant to succinic acid or malic acid production

• Contributing to a systems-level understanding of how protein synthesis capacity influences metabolic flux

This knowledge could complement existing metabolic engineering strategies for M. succiniciproducens, such as those using elementary mode analysis to identify targets for improving succinic acid production or approaches using dimethylsulfoxide as an electron acceptor for malic acid production .