Recombinant Treponema denticola 50S ribosomal protein L18 (rplR)

What is the function of 50S ribosomal protein L18 in bacterial cells?

The 50S ribosomal protein L18 is one of the three 5S rRNA-binding ribosomal proteins (along with L5 and L25) that form an integral part of the ribosomal complex. L18 is essential for cell viability in most bacteria, including E. coli. It plays a critical role in the incorporation of 5S rRNA into the 50S ribosomal subunit, which is necessary for the assembly of functional ribosomes capable of protein synthesis . Studies have demonstrated that L18 is indispensable for the in vivo assembly of active ribosomes, and without it, the translational machinery cannot function properly, leading to cellular death .

How conserved is L18 across bacterial species?

L18 is highly conserved across all three domains of life (Bacteria, Archaea, and Eukarya), suggesting its fundamental importance in ribosomal function . In bacteria, homologs of E. coli L5 and L18 are found in virtually all species, underscoring their essential roles in ribosome assembly and protein synthesis. The conservation of L18 across diverse bacterial species, including T. denticola, indicates its evolutionary significance and functional necessity in the translational machinery .

What is the structural relationship between L18 and 5S rRNA?

The L18 protein forms specific binding interactions with 5S rRNA, contributing to the formation of an autonomous structural domain within the ribosome. This protein-RNA complex is essential for ribosome function. The binding of L18 to 5S rRNA is a prerequisite for the proper incorporation of 5S rRNA into the 50S ribosomal subunit . The L18-5S rRNA interaction helps stabilize the tertiary structure of the ribosome and may facilitate communication between the peptidyl transferase and GTPase centers of the ribosome through the 5S rRNA, which has been suggested to link these two functional centers .

How does the absence of L18 affect ribosomal assembly and function in different bacterial species?

The absence of L18 has profound effects on ribosomal assembly and function. In E. coli, knockout of the rplR gene is lethal, indicating that L18 is essential for cell viability . Experiments have shown that ribosomes lacking 5S rRNA (which requires L18 for incorporation) are unable to synthesize polypeptides, retaining only EF-G-dependent GTPase activity . The essentiality of L18 is likely due to its role in ensuring the correct incorporation of 5S rRNA into the 50S subunit, without which the ribosome cannot properly form or function. Unlike L25 (another 5S rRNA-binding protein) whose absence leads to slow growth but not lethality, the absence of L18 completely prevents viable ribosome formation .

What are the differences in binding characteristics between T. denticola L18 and other bacterial L18 proteins?

While specific comparative data on T. denticola L18 binding characteristics is limited in the provided search results, research on various bacterial L18 proteins suggests species-specific adaptations in binding affinity and specificity. These differences may relate to structural variations in the binding interface between L18 and 5S rRNA across bacterial species. Understanding these species-specific binding characteristics could provide insights into evolutionary adaptations of the translational machinery and potential targets for species-specific antimicrobial development.

How does post-translational modification affect the function of L18 in different bacterial contexts?

Post-translational modifications can significantly influence the binding affinity, stability, and functional properties of L18. These modifications might include phosphorylation, methylation, or acetylation, which could regulate the interaction between L18 and 5S rRNA or other ribosomal components. Species-specific post-translational modifications may represent adaptations to different environmental conditions or metabolic states. Research examining how these modifications differ between T. denticola and other bacterial species could reveal mechanisms of translational regulation specific to different bacterial lifestyles and ecological niches.

What are the optimal conditions for expressing recombinant T. denticola L18 in heterologous systems?

The expression of recombinant T. denticola L18 can be achieved in various heterologous systems, including E. coli, yeast, baculovirus, or mammalian cell expression systems . For E. coli expression, which is commonly used due to its simplicity and high yield, optimal conditions typically include:

• Vector selection: pET series vectors with T7 promoters often provide good expression levels

• Host strain: BL21(DE3) or its derivatives are frequently used for ribosomal protein expression

• Induction conditions: 0.5-1.0 mM IPTG at OD600 0.6-0.8

• Temperature: Lower temperatures (16-25°C) often improve solubility

• Growth media: Enriched media such as TB or 2xYT can enhance yield

For purification, a combination of affinity chromatography (using His-tag or other fusion tags) followed by ion-exchange and size-exclusion chromatography typically yields protein with ≥85% purity .

What methods are most effective for studying the interaction between recombinant L18 and 5S rRNA?

Several complementary approaches can be used to study L18-5S rRNA interactions:

• Electrophoretic Mobility Shift Assays (EMSA): To assess binding affinity and specificity

• Surface Plasmon Resonance (SPR): For real-time binding kinetics analysis

• Isothermal Titration Calorimetry (ITC): To determine thermodynamic parameters of binding

• UV crosslinking and footprinting: To identify specific nucleotides involved in the interaction

• Cryo-EM or X-ray crystallography: For high-resolution structural analysis of the complex

These methods can be combined to provide comprehensive characterization of the interaction. For instance, EMSA can be used for initial screening of binding conditions, followed by SPR or ITC for quantitative analysis of binding parameters, and structural studies for atomic-level details of the interaction interface.

How can researchers verify the functional integrity of recombinant T. denticola L18?

Verification of functional integrity for recombinant T. denticola L18 can be assessed through multiple approaches:

• 5S rRNA binding assay: Testing the ability of the recombinant protein to bind 5S rRNA using EMSA or filter-binding assays

• In vitro reconstitution: Assessing the incorporation of recombinant L18 into 50S ribosomal subunits

• Complementation studies: Testing whether the recombinant protein can restore growth in L18-depleted systems

• Structural analysis: Circular dichroism (CD) spectroscopy to confirm proper protein folding

• Translation assays: Evaluating the impact of the recombinant protein on in vitro translation systems

A combination of these methods provides comprehensive validation of the functional integrity of the recombinant protein.

How should researchers interpret differences in ribosomal function when comparing wild-type and recombinant L18 in translation assays?

When interpreting differences in ribosomal function between wild-type and recombinant L18 in translation assays, researchers should consider several factors:

Based on studies with L25-defective ribosomes, differences in translation of natural mRNA without changes in poly(U) translation might indicate defects in specific translation steps such as initiation or ribosome recycling rather than in the elongation process itself .

What analytical approaches can distinguish between defects in ribosome assembly versus functional defects in assembled ribosomes?

Distinguishing between assembly defects and functional defects in assembled ribosomes requires a multi-faceted analytical approach:

• Ribosome profiling: Sucrose density gradient analysis can reveal abnormalities in subunit ratios or 70S formation, indicating assembly defects

• Protein composition analysis: Two-dimensional gel electrophoresis can identify missing or substoichiometric ribosomal proteins

• rRNA analysis: Northern blotting or primer extension can detect processing or structural defects in ribosomal RNA

• Functional assays with isolated fractions: Testing specific activities (GTPase, peptidyl transferase) of purified ribosomal fractions

• Comparative translation assays: Using different templates (poly(U) vs. natural mRNA) to pinpoint specific functional defects

For example, in studies of L25-defective ribosomes, normal sedimentation profiles and protein composition (except for the absence of L25) indicated proper assembly, while defects in natural mRNA translation but not poly(U) translation suggested specific functional impairments rather than general assembly problems .

How can researchers differentiate the effects of L18 mutations on various steps of protein synthesis?

Researchers can differentiate the effects of L18 mutations on various steps of protein synthesis through specialized assays that isolate each translation step:

By comparing results across these assays, researchers can pinpoint which specific translation step is affected by L18 mutations. For instance, if mutations impact natural mRNA translation but not poly(U) translation (as observed with L25-defective ribosomes), this suggests defects in initiation, termination, or recycling rather than elongation . Similarities to phenotypes observed with RRF mutations might indicate specific defects in the ribosome recycling step .

How do the structural and functional properties of T. denticola L18 compare with those of other oral pathogens?

Comparing T. denticola L18 with L18 proteins from other oral pathogens can provide insights into species-specific adaptations and potential targets for intervention. While detailed comparative data specific to T. denticola is not provided in the search results, general patterns can be inferred from studies of ribosomal proteins across bacterial species.

The L18 protein is highly conserved in terms of its core functional domains across bacterial species, but may exhibit species-specific variations in surface residues or regulatory regions. These variations could influence interactions with other ribosomal components or response to environmental conditions relevant to the oral cavity. Comparative structural and functional analyses could reveal adaptations specific to the lifestyle of T. denticola as an oral spirochete compared to other oral bacteria with different ecological niches or virulence properties.

What is known about the role of L18 in antibiotic resistance mechanisms across different bacterial species?

The ribosome is a common target for antibiotics, and variations in ribosomal proteins, including L18, can contribute to antibiotic resistance mechanisms. Research comparing L18 sequences and structures across antibiotic-resistant and susceptible strains could identify mutations associated with resistance phenotypes. Since L18 interacts with 5S rRNA and is essential for ribosome assembly, modifications in this protein might alter the binding of antibiotics that target the large ribosomal subunit.

Understanding species-specific variations in L18 structure and function could potentially inform the development of targeted antimicrobial strategies against T. denticola and other oral pathogens, possibly exploiting unique features of their translational machinery.

What are the most promising research directions for studying T. denticola L18 in the context of oral microbiome dynamics?

Future research on T. denticola L18 could productively focus on several areas:

• Host-pathogen interactions: Investigating whether L18 plays roles beyond translation in host-pathogen interactions, potentially through moonlighting functions when expressed on the bacterial surface or released during infection

• Microbiome interactions: Examining how variations in ribosomal proteins, including L18, might influence competitive fitness within the complex oral microbiome

• Stress adaptation: Analyzing how T. denticola L18 contributes to adaptation to the stressful environment of periodontal pockets, potentially drawing parallels to the stress-related functions observed in homologous proteins like the CTC protein in B. subtilis

• Targeted antimicrobials: Exploring whether species-specific features of T. denticola L18 could be exploited for selective antimicrobial development

These directions could enhance our understanding of T. denticola pathogenesis and potentially reveal novel therapeutic approaches for periodontal disease.

How might advanced structural biology techniques enhance our understanding of L18 function in T. denticola?

Advanced structural biology techniques, including cryo-electron microscopy, X-ray crystallography, and NMR spectroscopy, could provide detailed insights into T. denticola L18 structure and function. These approaches could:

• Resolve atomic-level details of T. denticola L18 interactions with 5S rRNA and other ribosomal components

• Identify species-specific structural features that might influence ribosome assembly or function

• Visualize conformational changes during different stages of translation

• Provide templates for structure-based drug design targeting unique features of T. denticola ribosomes