Recombinant Pinus koraiensis 30S ribosomal protein S7, chloroplastic (rps7)

What is the function of chloroplastic ribosomal protein S7 in Pinus koraiensis?

The 30S ribosomal protein S7 (rps7) in Pinus koraiensis chloroplasts plays a critical role in the small ribosomal subunit assembly and function. Based on studies of ribosomal proteins across species, rps7 is involved in the formation of the exit channel through which mRNA passes during translation. The protein connects the head of the 30S subunit to the platform in the E site of the ribosome, helping maintain the structural integrity necessary for proper translation . In chloroplasts specifically, rps7 contributes to organellar protein synthesis and may be involved in chloroplast gene expression regulation, which is essential for photosynthetic function in conifer species like Korean pine.

What are the optimal methods for expressing recombinant P. koraiensis rps7 protein in E. coli systems?

For expressing recombinant P. koraiensis rps7 in E. coli, researchers should consider the following methodological approach:

• Vector selection: Use pET expression vectors with T7 promoter systems for high-level expression.

• Codon optimization: Optimize codons for E. coli expression, as plant chloroplast genes may contain codons rarely used in E. coli.

• Expression conditions:

  • Initial induction with 0.1-0.5 mM IPTG

  • Growth temperature of 18-25°C after induction to minimize inclusion body formation

  • Expression in BL21(DE3) or Rosetta strains to address potential rare codon issues

• Purification strategy:

  • Include a His6-tag for affinity purification

  • Use buffer containing 20 mM Tris-HCl (pH 8.0), 100-300 mM NaCl, and 1 mM DTT

  • Consider adding glycerol (10-30%) for stability

Potential challenges include protein insolubility and proper folding. Addition of chaperones or fusion partners (SUMO, MBP, or GST) may improve solubility and folding of the recombinant protein.

How can I design experiments to investigate the interaction between P. koraiensis rps7 and other ribosomal proteins?

To investigate interactions between P. koraiensis rps7 and other ribosomal proteins, consider these methodological approaches:

In vitro approaches:

• Co-immunoprecipitation (Co-IP): Express recombinant rps7 with epitope tags and potential interacting partners to identify physical interactions.

• Pull-down assays: Use purified His-tagged rps7 as bait to capture interacting proteins from chloroplast extracts.

• Surface Plasmon Resonance (SPR): Determine binding kinetics and affinity constants between rps7 and other proteins such as S11.

In vivo approaches:

• Yeast two-hybrid (Y2H): Screen for protein-protein interactions using rps7 as bait.

• Bimolecular Fluorescence Complementation (BiFC): Visualize interactions in plant cells.

Structural approaches:

• Cryo-EM analysis: Determine the structure of the P. koraiensis chloroplast ribosome to visualize rps7 interactions.

• Cross-linking coupled with mass spectrometry: Identify amino acid residues involved in interactions.

Based on previous studies of ribosomal proteins, focus particularly on the interaction between rps7 and rps11, as this interaction has been shown to be functionally significant in the E site and affects translational fidelity .

How does P. koraiensis rps7 differ functionally from rps7 proteins in other plant species?

Functional differences between P. koraiensis rps7 and other plant rps7 proteins likely stem from evolutionary adaptations to environmental conditions. While the core function remains conserved, subtle differences may exist:

• Sequence variations: Comparative sequence analysis reveals that while the functional domains are highly conserved, specific residues may vary between species, potentially affecting protein-protein interactions or RNA binding specificity.

• Thermal stability: As P. koraiensis is adapted to cooler temperate regions, its rps7 may have evolved enhanced stability at lower temperatures compared to rps7 from tropical plants.

• RNA binding properties: Studies of ribosomal proteins suggest that species-specific differences in rps7 may affect mRNA binding capacity and translational fidelity . The P. koraiensis rps7 may have unique RNA binding characteristics adapted to regulate translation of chloroplast genes important for cold tolerance.

• Regulatory mechanisms: The regulation of rps7 expression may differ in P. koraiensis, particularly in the 5' UTR region, which has been shown to affect gene expression in other species .

A comprehensive functional comparison would require expressing recombinant rps7 proteins from multiple species and conducting comparative binding assays, structural studies, and in vitro translation experiments.

What role does rps7 play in stress response mechanisms in P. koraiensis chloroplasts?

The role of rps7 in stress response mechanisms in P. koraiensis chloroplasts can be inferred from studies on light stress and other environmental conditions in related species:

• Light stress response: Transcriptomic analyses of Pinus koraiensis under different light conditions reveal complex regulatory networks involving chloroplast functions . While rps7 was not specifically mentioned, chloroplast ribosomal proteins like rps7 are likely involved in translational regulation of stress-responsive proteins.

• Cold tolerance mechanisms: As P. koraiensis is adapted to cold environments, chloroplast translation machinery including rps7 may be specialized for maintaining protein synthesis under low-temperature conditions.

• Oxidative stress: Under stress conditions that increase reactive oxygen species (ROS) in chloroplasts, ribosomal proteins may play protective roles. Korean pine extracts have been shown to increase superoxide dismutase (SOD) activity , suggesting coordinated responses involving chloroplast functions.

Experimental approaches to study rps7's role in stress response could include:

• Analysis of rps7 expression levels under various stress conditions

• Creation of transgenic plants with modified rps7 expression

• Proteomics analysis of chloroplast ribosomes under stress conditions

How has the rps7 gene evolved across different Pinus species, and what insights does this provide about P. koraiensis adaptation?

Evolutionary analysis of the rps7 gene across Pinus species reveals patterns of conservation and adaptation:

• Sequence conservation: Comparative genomic studies of pine species indicate that ribosomal proteins like rps7 are generally highly conserved due to their essential function. A study of chloroplast genomes estimated that they evolved from common ancestors approximately 1293 million years ago .

• Selection pressure: The rps7 gene likely experiences purifying selection, maintaining its functional domains while allowing some variation in less critical regions.

• Geographical patterns: Genetic studies of P. koraiensis across its range show patterns of variation related to geographical distribution:

This north-south gradient in genetic diversity supports the hypothesis of northward migration of Korean pine .

• Population structure: Analysis of P. koraiensis populations reveals genetic differentiation influenced by geographical barriers. For example, the Xiaoxing'anling mountains appear to function as a barrier against gene flow between certain Chinese populations .

To specifically study rps7 evolution, researchers should sequence this gene across multiple pine populations and apply phylogenetic methods to identify adaptive mutations and selection pressures.

What can comparative analysis of chloroplast rps7 genes tell us about the evolutionary history of conifers?

Comparative analysis of chloroplast rps7 genes provides valuable insights into conifer evolutionary history:

• Phylogenetic relationships: Molecular clock analysis of chloroplast genes, including rps7, can help resolve relationships among closely related pine species. For example, studies have shown that P. koraiensis and P. griffithii diverged from P. armandii and P. pumila approximately 1.37 million years ago .

• Genome architecture evolution: Analysis of the location and context of rps7 within the chloroplast genome across species reveals patterns of chloroplast genome rearrangements and evolution. In Pinaceae, the typical highly reduced inverted repeats (IRs) represent a derived state .

• Gene loss patterns: While rps7 is generally conserved in photosynthetic plants, patterns of gene loss in the chloroplast genome vary across lineages. Studies have identified specific signature genes that have been lost in certain lineages, providing markers for evolutionary events .

• Adaptive evolution: By analyzing the ratio of nonsynonymous to synonymous substitutions (dN/dS) in rps7 sequences across species, researchers can identify signatures of selection that may correlate with adaptation to different environments.

For a comprehensive study, researchers should combine chloroplast gene sequences with nuclear markers to address potential incongruence between organellar and nuclear evolutionary histories, as has been observed in some pine species .

What are common challenges in isolating and purifying recombinant P. koraiensis rps7, and how can they be addressed?

Researchers face several challenges when isolating and purifying recombinant P. koraiensis rps7:

• Protein solubility issues: Ribosomal proteins often form inclusion bodies when overexpressed.

  • Solution: Lower induction temperature (16-18°C), reduce IPTG concentration (0.1-0.2 mM), or use fusion tags like MBP or SUMO.

• Protein stability concerns: Purified rps7 may be prone to degradation or aggregation.

  • Solution: Include protease inhibitors throughout purification, add stabilizing agents (10-30% glycerol, 1-5 mM DTT), and maintain cold temperatures during handling .

• Contaminant RNA binding: As an RNA-binding protein, rps7 may co-purify with bacterial RNA.

  • Solution: Treat lysates with RNase or include high salt washes (500 mM NaCl) during purification.

• Proper folding assessment: Ensuring the recombinant protein adopts its native conformation.

  • Solution: Use circular dichroism spectroscopy to assess secondary structure, and functional assays to verify RNA-binding activity.

• Low expression yield: Plant chloroplast proteins may express poorly in bacterial systems.

  • Solution: Optimize codons for E. coli, use strong promoters, and try different E. coli strains (Rosetta, Arctic Express).

Based on protocols for other recombinant ribosomal proteins, a recommended purification buffer would contain 20 mM Tris-HCl (pH 8.0), 100-300 mM NaCl, 1 mM DTT, and 10% glycerol .

How can researchers verify the functional integrity of recombinant P. koraiensis rps7 protein?

To verify the functional integrity of recombinant P. koraiensis rps7 protein, researchers should employ multiple complementary approaches:

• Structural analysis:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure elements

  • Limited proteolysis to assess proper folding

  • Thermal shift assays to determine stability

• RNA binding assays:

  • Electrophoretic mobility shift assays (EMSA) with rRNA targets

  • Filter-binding assays to quantify binding affinity

  • Surface plasmon resonance (SPR) to measure binding kinetics

• Protein-protein interaction verification:

  • Pull-down assays with other ribosomal proteins, particularly S11

  • Size exclusion chromatography to assess complex formation

• Functional assays:

  • In vitro reconstitution assays with other ribosomal components

  • 30S subunit assembly assays

  • Translation fidelity assessment in reconstituted systems

• Complementation studies:

  • Bacterial complementation assays in S7-deficient strains

  • Chloroplast transformation in model plants to assess in vivo function

A positive control using a well-characterized ribosomal protein S7 from another species (such as E. coli) should be included in these assays for comparison. Researchers should note that functional verification requires multiple lines of evidence, as structural integrity alone does not guarantee functional activity.

How can recombinant P. koraiensis rps7 be used to study chloroplast translation mechanisms under environmental stress?

Recombinant P. koraiensis rps7 offers several research applications for studying chloroplast translation under environmental stress:

• In vitro translation systems:

  • Develop reconstituted chloroplast translation systems incorporating recombinant rps7

  • Compare translation efficiency under simulated stress conditions (temperature variation, altered ionic conditions, oxidative stress)

  • Assess how rps7 mutations affect translation of stress-response genes

• Structure-function analysis:

  • Create site-directed mutants of rps7 to identify residues important for stress adaptation

  • Compare P. koraiensis rps7 with orthologues from species adapted to different environments

  • Study how environmental stressors affect rps7-RNA interactions

• Stress response pathway integration:

  • Investigate how rps7 interacts with stress-responsive factors in chloroplasts

  • Study the relationship between light stress response networks and chloroplast translation

  • Examine how translation regulation contributes to adaptation mechanisms

• Molecular evolution studies:

  • Compare sequences and functional properties of rps7 from P. koraiensis populations from different environments

  • Identify potential adaptive mutations in rps7 related to environmental stress tolerance

  • Reconstruct the evolutionary history of stress adaptation in pine species

This research is particularly relevant since P. koraiensis has evolved mechanisms to cope with various environmental stressors, and chloroplast translation plays a key role in stress response by regulating the synthesis of proteins involved in photosynthesis and reactive oxygen species management.

What insights can the study of P. koraiensis rps7 provide for understanding ribosomal protein function in cancer and other human diseases?

While P. koraiensis rps7 is a chloroplast protein not found in humans, comparative studies can provide valuable insights into fundamental mechanisms relevant to human ribosomal protein functions and disease:

• Conserved structural motifs: Comparative analysis of plant and human ribosomal proteins can identify highly conserved structural elements essential for function. Human RPS7 has been implicated in cancer progression, with contradictory findings in different cancers:

• RNA-binding mechanisms: The RNA-binding domain of rps7 is evolutionarily conserved. Insights from P. koraiensis rps7-RNA interactions could inform understanding of human RPS7's role in mRNA stabilization, as seen in hepatocellular carcinoma where RPS7 stabilizes LOXL2 mRNA .

• Protein-protein interactions: The interaction between rps7 and rps11 affects translational fidelity . Similar interactions in human ribosomes might influence cancer progression through translation quality control.

• Translational regulation: Understanding how plant rps7 regulates translation under stress could provide concepts applicable to stress response in human cells, potentially relevant to cancer where translational dysregulation is common.

While direct application of plant rps7 findings to human disease requires caution due to evolutionary distance, the fundamental principles of ribosome function and regulation discovered in diverse systems often have parallel implications in human biology.

How might CRISPR-Cas9 technologies be applied to study the function of chloroplastic rps7 in P. koraiensis?

Applying CRISPR-Cas9 technologies to study chloroplastic rps7 in P. koraiensis presents unique challenges and opportunities:

• Technical approach for chloroplast genome editing:

  • Traditional nuclear CRISPR systems must be modified to target chloroplasts

  • Chloroplast-targeted CRISPR requires specialized transit peptides

  • Biolistic transformation is typically required for chloroplast transformation in conifers

• Experimental design strategies:

  • Create knock-down rather than knock-out mutations (as rps7 is likely essential)

  • Design sgRNAs targeting non-conserved regions to create partial function mutants

  • Create site-specific mutations to study structure-function relationships

• Alternative approaches for P. koraiensis:

  • Use model plant systems first (tobacco, Arabidopsis) for proof-of-concept

  • Apply transplastomic approaches to replace native rps7 with modified versions

  • Use transient expression systems to express mutant rps7 versions

• Phenotypic analyses:

  • Assess effects on chloroplast translation efficiency

  • Measure photosynthetic parameters under various environmental conditions

  • Analyze stress responses in modified plants

• Validation methods:

  • RT-qPCR to confirm changes in rps7 expression

  • Western blotting to assess protein levels

  • Ribosome profiling to evaluate translation efficiency

While CRISPR-Cas9 editing of conifer chloroplasts remains technically challenging, recent advances in plastid transformation technologies make this an emerging frontier for studying genes like rps7 in their native context.

How can systems biology approaches integrate transcriptomic and metabolomic data to understand the broader role of rps7 in P. koraiensis physiology?

Systems biology approaches can reveal the broader physiological context of rps7 function in P. koraiensis:

• Multi-omics data integration framework:

  • Combine transcriptomics, proteomics, and metabolomics data under various conditions

  • Use chloroplast ribosome profiling to assess translation efficiency genome-wide

  • Apply network analysis to identify gene modules co-regulated with rps7

• Environmental response network mapping:

  • Study how light stress affects translation of photosynthetic genes

  • Compare metabolic profiles under different light conditions in relation to chloroplast translation efficiency

  • Identify metabolic pathways most sensitive to alterations in chloroplast translation

• Methodological approach:

  • Generate condition-specific datasets (various light, temperature, nutrient conditions)

  • Use weighted gene co-expression network analysis (WGCNA) to identify functional modules

  • Apply flux balance analysis to model metabolic consequences of altered translation

• Integration with phenotypic data:

  • Connect molecular networks to physiological parameters (photosynthetic efficiency, growth rates)

  • Assess how genetic variation in rps7 correlates with phenotypic differences among populations

  • Model how environmental adaptations are reflected in translation regulation

This approach would build on existing transcriptomic and metabolomic studies of P. koraiensis under light stress, which have identified significantly enriched pathways including flavonoid biosynthesis, phenylpropanoid biosynthesis, and plant hormone signal transduction . The role of chloroplast translation in coordinating these responses remains an important area for investigation.

How do we reconcile contradictory findings about the role of ribosomal protein S7 in different biological systems?

Contradictory findings about ribosomal protein S7 across different biological systems can be reconciled through several analytical approaches:

• Comparative analysis framework:

  • Distinguish between cytosolic (eukaryotic) RPS7 and chloroplastic (prokaryotic-like) rps7

  • Recognize that despite sequence homology, these proteins function in distinct cellular compartments

  • Consider evolutionary divergence in function despite structural conservation

• Context-dependent functions:

  • In human cancers, RPS7 shows tissue-specific effects—tumor suppressive in colorectal cancer but oncogenic in hepatocellular and prostate cancers

  • In plants, rps7 functions primarily in chloroplast translation but may have acquired secondary roles

• Methodological considerations:

  • Different experimental approaches (in vitro vs. in vivo, overexpression vs. knockdown)

  • Variation in cell/tissue types and model organisms used

  • Differences in environmental or experimental conditions

• Reconciliation strategies:

  • Design experiments that directly compare S7 function across systems under identical conditions

  • Use domain-swapping experiments to identify functional regions responsible for different activities

  • Perform comprehensive interactome studies to identify context-specific protein partners

Specific contradictions in rps7 literature can be addressed through carefully designed comparative studies that account for the unique aspects of each biological system while focusing on conserved mechanisms.

What experimental approaches can address conflicting data about P. koraiensis genetics across different regional populations?

To address conflicting data about P. koraiensis genetics across different regional populations, researchers should implement the following experimental approaches:

• Standardized sampling and analysis protocols:

  • Develop consistent sampling strategies across the entire species range

  • Standardize DNA extraction and sequencing methodologies

  • Apply uniform bioinformatic pipelines for data analysis

• Comprehensive genetic marker selection:

  • Combine multiple marker types (chloroplast, mitochondrial, and nuclear)

  • Include both coding regions (like rps7) and non-coding regions

  • Use genome-wide approaches (e.g., RAD-seq) to capture broader genetic variation

• Statistical frameworks for reconciling discrepancies:

  • Apply meta-analysis techniques to integrate data from different studies

  • Use Bayesian approaches to account for sampling uncertainty

  • Implement multivariate statistical methods to identify patterns across datasets

• Environmental correlation analysis:

  • Integrate genetic data with detailed environmental parameters

  • Apply landscape genetics approaches to identify environmental drivers of genetic structure

  • Develop environmental association models to test adaptive hypotheses