Recombinant Mannheimia succiniciproducens 50S ribosomal protein L3 (rplC)

What is the structural and functional role of rplC in bacterial ribosomes?

The 50S ribosomal protein L3 is one of the primary rRNA binding proteins in bacterial ribosomes. It binds directly near the 3'-end of the 23S rRNA, where it nucleates assembly of the 50S subunit . L3 is an essential and indispensable component for formation of the peptidyl transferase center (PTC) . Studies demonstrate that L3 is one of the first ribosomal proteins to be assembled onto the 23S rRNA and is among only two proteins (along with L24) capable of initiating assembly of the 50S ribosomal subunit . Its positioning is critical - while the main part of L3 is located on the surface of the 50S ribosomal subunit, it features a branched loop that extends close to the PTC, which serves as the binding site for many different ribosomal antibiotics .

How conserved is the rplC gene across bacterial species?

The rplC gene belongs to the universal ribosomal protein uL3 family, indicating high conservation across bacterial species . This conservation reflects its fundamental role in ribosome function. The gene is located in the S10 operon in bacterial genomes (as observed in E. coli), which encodes 11 ribosomal proteins and is strongly regulated by the L4 ribosomal protein . Despite this conservation, specific mutations in the L3 sequence, particularly in loops near the PTC, can be found in different bacterial strains and may confer differential antibiotic susceptibility profiles . Comparative analysis of L3 sequences across species can provide insights into evolutionary adaptations related to ribosome function and antibiotic interactions.

What experimental techniques are most appropriate for initial characterization of recombinant rplC?

Initial characterization of recombinant rplC should employ multiple complementary techniques:

• SDS-PAGE and Western blotting: Using antibodies against conserved L3 peptide regions to verify expression and protein stability .

• Mass spectrometry: Confirming protein identity through peptide mass fingerprinting following trypsin digestion of excised gel bands .

• Circular dichroism (CD) spectroscopy: Assessing secondary structure elements to ensure proper folding.

• Size exclusion chromatography: Evaluating oligomeric state and homogeneity of the purified protein.

• RNA binding assays: Testing the ability of recombinant rplC to bind 23S rRNA fragments to confirm functional activity.

These methods provide a comprehensive initial assessment of recombinant rplC before proceeding to more complex functional studies.

What expression systems are most effective for producing recombinant M. succiniciproducens rplC?

Based on established protocols for ribosomal proteins, several expression systems can be considered for M. succiniciproducens rplC:

Table 1: Comparison of Expression Systems for Recombinant rplC Production

The E. coli expression system has been successfully used for studies involving ribosomal protein L3, as demonstrated in plasmid-based complementation studies . Expression should be verified using Western blotting with antibodies targeting conserved epitopes of L3 . For optimal expression, the gene sequence may need codon optimization for the chosen expression host.

What purification protocol yields high-purity recombinant rplC suitable for structural studies?

A multi-step purification protocol is recommended:

• Initial clarification: Centrifugation of cell lysate (30,000 × g, 30 min, 4°C) followed by filtration.

• Affinity chromatography: Use of polyhistidine tags with IMAC purification on Ni-NTA resin, with stepwise imidazole elution (50 mM, 100 mM, 250 mM, 500 mM).

• Ion exchange chromatography: Resource Q or S columns (depending on the calculated pI of M. succiniciproducens rplC) to remove nucleic acid contamination.

• Size exclusion chromatography: Superdex 75 or Superdex 200 columns to achieve >95% purity.

• Tag removal: If structural studies are planned, consider TEV or PreScission protease cleavage of affinity tags.

Protein purity should be assessed at each step using SDS-PAGE, and final verification can be performed using mass spectrometry, as demonstrated in studies of L3 mutations .

How can researchers assess the functional integrity of purified recombinant rplC?

Functional integrity assessment should include:

• 23S rRNA binding assays: Electrophoretic mobility shift assays (EMSA) or surface plasmon resonance (SPR) to quantify binding to 23S rRNA fragments.

• In vitro ribosome assembly: Testing the ability of the recombinant protein to participate in ribosomal subunit reconstitution.

• Thermal stability analysis: Differential scanning fluorimetry to assess protein stability and proper folding.

• Peptidyl transferase activity assays: When incorporated into reconstituted ribosomes, functional rplC should support peptidyl transferase activity, which can be measured using standard assays.

The functional integrity is critical as L3 is essential for bacterial viability and plays a key role in ribosome assembly .

What techniques are most effective for studying rplC-rRNA interactions at the molecular level?

Multiple complementary approaches provide insights into rplC-rRNA interactions:

• X-ray crystallography: For high-resolution structural information when co-crystallized with rRNA fragments.

• Cryo-electron microscopy: Increasingly used for visualizing ribonucleoprotein complexes at near-atomic resolution.

• Nuclear magnetic resonance (NMR): For studying dynamics of specific domains, particularly the branched loop that extends to the PTC.

• Cross-linking mass spectrometry: To identify specific contact points between rplC and rRNA.

• Hydrogen-deuterium exchange mass spectrometry: For mapping interaction interfaces and conformational changes.

• Computational modeling: As used in studies of L3 mutations, computational approaches can model structural changes and interactions .

These methods collectively provide a comprehensive understanding of how rplC interacts with rRNA to facilitate ribosome assembly and function.

How does the branched loop of rplC contribute to peptidyl transferase center formation?

The branched loop of the L3 protein extends close to the peptidyl transferase center (PTC) . This positioning is critical because:

• L3, along with L2 and L4, modulates peptidyl transferase activity by stabilizing and maintaining the conformation of rRNA at the active site .

• Mutations in this loop region can affect peptidyl transferase activity, as demonstrated in both prokaryotic and eukaryotic studies .

• The loop contributes to the binding pocket for PTC-targeting antibiotics, explaining why L3 mutations can confer antibiotic resistance .

• The positioning of the loop influences the conformation of key nucleotides in the PTC, affecting tRNA binding at A and P sites .

Computational modeling studies have shown that mutations in the L3 protein can cause structural changes that affect antibiotic binding to the PTC, further highlighting the critical role of the branched loop .

What methods can be used to study the effects of specific mutations in M. succiniciproducens rplC?

To study effects of specific rplC mutations, researchers can employ:

• Plasmid exchange systems: As demonstrated in E. coli studies, constructing a chromosomal L3 deletion strain with essential L3 gene provided on plasmids allows replacement of wild-type with mutated L3 genes . This enables investigation of the effect of single mutations without wild-type L3 background interference.

• Site-directed mutagenesis: Standard overlap extension PCR can be used to introduce specific mutations into the L3 gene .

• Growth kinetics analysis: Measuring doubling times of mutant strains to assess fitness effects of mutations .

• Antibiotic susceptibility testing: Determining MICs for various antibiotics to identify resistance phenotypes .

• Computational modeling: Assessing the impact of L3 mutations on 50S structure and antibiotic binding using molecular dynamics simulations and docking studies .

These approaches provide complementary data on structural, functional, and phenotypic effects of L3 mutations.

How do specific mutations in rplC affect susceptibility to peptidyl transferase center-targeting antibiotics?

L3 mutations can significantly alter antibiotic susceptibility profiles:

Table 2: Effect of L3 Mutations on Antibiotic Susceptibility

Different mutations in the L3 protein, particularly those in the loops near the PTC, have varying effects on antibiotic susceptibility . Some mutations may reduce susceptibility to linezolid (an oxazolidinone), while others affect tiamulin (a pleuromutilin) susceptibility . The mechanism involves alterations in the binding pocket for these antibiotics, either through direct steric hindrance or indirect perturbation of key rRNA nucleotides such as G2505 and U2506 of 23S rRNA .

Importantly, mutations in L3 may come with fitness costs, as seen with the G144D mutation that caused significant growth inhibition with a doubling time of 130 minutes in E. coli . The impact of specific mutations can vary between bacterial species, explaining why some mutations are well-tolerated in one species but detrimental in others .

What computational approaches are valuable for predicting the impact of novel rplC mutations?

Computational approaches for studying rplC mutations include:

• Docking simulations: Using methodologies like XP Glide to quantify binding mode and energy of antibiotic binding to the 50S ribosomal subunit . This provides a glide score that reflects binding strength and predicts positioning of the antibiotic at the binding site.

• Cluster modeling: Creating sphere models of atoms around the antibiotic binding site as an efficient representation of the receptor for calculations .

• Structural analysis: Imposing mutations into the 50S structure and analyzing local conformational changes.

• Molecular dynamics simulations: Assessing stability and dynamic changes in ribosome structure resulting from L3 mutations.

• Quantum mechanical calculations: For detailed analysis of electron distribution and bonding changes at the PTC.

These approaches, while highly parameterized, provide valuable insights into potential structural and functional impacts of mutations before experimental validation .

How can recombinant rplC be used in ribosome reconstitution experiments?

Ribosome reconstitution experiments using recombinant rplC provide powerful insights into ribosome assembly and function:

• In vitro assembly systems: Taking advantage of L3's role as one of the first proteins to be assembled onto 23S rRNA and its capability to initiate assembly of the 50S subunit .

• Incorporation of modified rplC: Introducing fluorescently labeled or chemically modified rplC to study assembly dynamics or create functional ribosomes with novel properties.

• Minimal ribosome systems: Determining the minimal set of components needed for PTC formation, given that L3 (along with L2 and L4) is essential for PTC formation .

• Hybrid ribosomes: Creating chimeric ribosomes with components from different species to study species-specific aspects of translation.

• Time-resolved assembly studies: Monitoring the kinetics of 50S subunit formation in the presence of wild-type versus mutant rplC.

These experiments capitalize on L3's essential role in ribosome assembly and can provide fundamental insights into this complex macromolecular machinery.

What are the implications of rplC research for developing novel antimicrobial strategies?

Research on rplC has significant implications for antimicrobial development:

• New target sites: Understanding the detailed structure and function of L3 and its interaction with the PTC can reveal novel binding pockets for antibiotic development.

• Resistance prediction: Knowledge of how L3 mutations confer resistance to existing antibiotics like linezolid and tiamulin allows for rational design of new compounds that maintain efficacy against resistant strains.

• Species-specific targeting: Exploiting subtle differences in L3 structure between pathogenic and commensal bacteria to develop narrow-spectrum antibiotics.

• Combination therapies: Developing drugs that can bind simultaneously to different sites affected by L3, reducing the likelihood of resistance development.

• Ribosome assembly inhibitors: Creating compounds that specifically interfere with L3's role in ribosome assembly rather than targeting the mature ribosome.

The critical role of L3 in ribosome function and its documented involvement in antibiotic resistance mechanisms make it a valuable focus for antimicrobial research .

What methodologies can assess the impact of rplC mutations on translational fidelity?

Several approaches can quantify how rplC mutations affect translational accuracy:

• Reporter systems: Using dual luciferase reporters with programmed frameshifting sites or stop codons to measure readthrough and frameshifting rates.

• Mass spectrometry: Analyzing the proteome of cells expressing mutant rplC to identify misincorporation events and quantify error rates.

• In vitro translation assays: Reconstituting ribosomes with wild-type or mutant rplC and measuring translation fidelity using defined mRNA templates.

• Single-molecule studies: Monitoring individual translation events to detect pausing, frameshifting, or other irregularities in real-time.

• Ribosome profiling: Genome-wide assessment of ribosome positioning and dynamics to identify translation anomalies associated with rplC mutations.

Studies in eukaryotic systems have already demonstrated that mutations in L3 can alter translational fidelity by changing frame-shifting efficiency , suggesting similar effects may occur in bacterial systems including M. succiniciproducens.