Recombinant Nelumbo lutea 30S ribosomal protein S7, chloroplastic (rps7)

What is the evolutionary relationship between Nelumbo lutea and Nelumbo nucifera, and how might this affect their rps7 proteins?

Nelumbo lutea (American lotus) and Nelumbo nucifera (Asian lotus) are the only two extant species in the Nelumbonaceae family. Comparative genomic analyses have revealed that these species diverged approximately 13.86 million years ago . Despite this divergence, they maintain high synteny (preservation of gene order) in their genomes without large chromosomal rearrangements .

How does the S7 protein interact with rRNA in the chloroplast ribosome?

The S7 protein plays a crucial role in the assembly and function of the 30S ribosomal subunit in chloroplasts. Based on structural studies of bacterial and chloroplast ribosomes, S7 interacts directly with 16S rRNA and initiates the assembly of the 30S subunit's head domain .

In studies of bacterial S7 (which shares homology with chloroplast S7), the protein recognizes specific sequence elements in 16S rRNA. These elements include regions in loop A (at the junction between helices 30 and 41) and loop B (proximal to helix 43) . Crystal structures of bacterial 30S subunits at 3.0 and 3.3 Å resolution have confirmed these interactions, showing that:

• The element in loop A interacts with residues in loop 2 and loop 5 of protein S7

• The element in loop B interacts with residues in the N-terminal region, loop 2, β-hairpin, and α-helix 4 of protein S7

Given the evolutionary conservation of ribosomal assembly, similar interaction patterns likely exist in the chloroplast S7 protein, although chloroplast-specific modifications may occur due to the unique architecture of the chloroplast ribosome .

What is the role of S7 in translational regulation in chloroplasts?

S7 protein appears to function as a translational regulator in chloroplasts, similar to its role in bacteria. In Chlamydomonas, S7 has been shown to bind to the 5' untranslated region (5'UTR) of the chloroplast rps7 gene . Specific mutations in this 5'UTR reduce expression of reporter genes, while second-site suppressors restore expression.

The regulatory functions of S7 involve:

• Binding to its own mRNA (autoregulation of translation)

• Potential binding to other chloroplast mRNAs

• Possible coordination of ribosome assembly with translation rates

Research has demonstrated that S7 protein can distinguish between different conformations of RNA and may play either a general or specific regulatory role in translation initiation in the chloroplast . The dual function of S7 in ribosome assembly and translational regulation highlights the complex integration of these processes in chloroplast gene expression.

What are the optimal conditions for reconstituting recombinant Nelumbo lutea rps7 protein for functional studies?

For optimal reconstitution of lyophilized recombinant Nelumbo lutea rps7 protein:

• Initial preparation: Briefly centrifuge the vial prior to opening to bring contents to the bottom.

• Reconstitution procedure:

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

  • Add glycerol to a final concentration of 5-50% (recommended default: 50%)

  • Aliquot for long-term storage at -20°C/-80°C

• Storage considerations:

  • The shelf life of liquid form is approximately 6 months at -20°C/-80°C

  • The shelf life of lyophilized form is approximately 12 months at -20°C/-80°C

  • Avoid repeated freezing and thawing

  • Store working aliquots at 4°C for up to one week

These recommendations are based on standard protocols for maintaining protein stability and activity for functional studies, but specific experimental conditions may require optimization.

What methods are most effective for studying rps7-RNA interactions in vitro?

Several complementary approaches have been successfully used to study ribosomal protein-RNA interactions, which can be applied to Nelumbo lutea rps7:

• Filter binding assays: These have been used to quantitatively measure the affinity of S7 for its RNA targets. In studies with bacterial S7, filter binding assays revealed that the same mutations that interfere with S7 binding to 16S rRNA also weaken its affinity for its mRNA, suggesting conserved binding mechanisms .

• Mutagenesis coupled with binding studies: Systematic mutation of specific residues in S7 (particularly in the N-terminal region, loop 2, and loop 5) followed by binding assays can identify critical amino acids for RNA interaction. For example, point mutations Q8A, F17G, K35Q, and M115G in bacterial S7 significantly decreased binding to both 16S rRNA and mRNA .

• RNA structure probing: Techniques such as hydroxyl radical footprinting, chemical probing, and crosslinking studies can map the interaction sites between S7 and its target RNAs.

• Cryo-electron microscopy: Recent advances in cryo-EM have enabled high-resolution structural determination of ribosomal complexes, allowing visualization of protein-RNA interactions in near-native states .

For in vitro studies specifically with recombinant Nelumbo lutea rps7, researchers should consider:

• Using physiologically relevant buffer conditions mimicking the chloroplast environment

• Including controls with known bacterial S7 for comparison

• Validating interactions with multiple complementary techniques

How can structural variations in rps7 across Nelumbo species inform our understanding of chloroplast ribosome evolution?

Studying structural variations in rps7 across Nelumbo species can provide valuable insights into chloroplast ribosome evolution:

• Comparative genomic analysis: The genomes of Nelumbo lutea and Nelumbo nucifera have been sequenced and revealed 29,533 structural variants (SVs) between them . Although specific SVs in rps7 were not highlighted in the available data, the methodologies used for genome-wide SV detection can be applied to analyze variations in this specific gene.

• Molecular clock analyses: The divergence time of 13.86 million years between Nelumbo species provides a temporal framework to estimate evolutionary rates of chloroplast ribosomal proteins. By comparing substitution rates in rps7 to those of other ribosomal proteins, researchers can identify conserved regions under purifying selection versus regions undergoing adaptive evolution.

• Functional implications: Specific amino acid substitutions in rps7 could affect:

  • RNA binding specificity or affinity

  • Interactions with other ribosomal proteins

  • Regulatory functions in translation

• Structural biology approaches: High-resolution structures of chloroplast ribosomes, like the 3.4 Å resolution structure of the spinach chloroplast 70S ribosome , provide templates for modeling species-specific variations in rps7 and predicting their functional consequences.

Comparing rps7 across Nelumbo species within the context of the complete chloroplast ribosome architecture could reveal adaptations specific to the different ecological niches of American and Asian lotus.

What are the challenges in expressing functional chloroplast rps7 in heterologous systems, and how can they be overcome?

Expressing functional chloroplast rps7 in heterologous systems presents several challenges:

• Codon usage bias: Chloroplast genes often have distinct codon usage patterns that differ from those of common expression hosts like E. coli. This can lead to:

  • Poor translation efficiency

  • Premature termination

  • Protein misfolding

  Solution: Optimize the coding sequence for the expression host while maintaining the amino acid sequence.

• Protein folding and solubility: As a ribosomal protein that normally functions in complex with rRNA and other proteins, isolated rps7 may have solubility issues.

  Solutions:

  • Express with solubility-enhancing tags (e.g., MBP, SUMO)

  • Use low-temperature induction to slow folding

  • Co-express with chaperones

  • Include stabilizing agents in purification buffers

• Functional assessment: Outside its native ribosomal context, assessing the functionality of recombinant rps7 can be challenging.

  Solutions:

  • In vitro reconstitution assays with chloroplast 16S rRNA

  • RNA binding assays using defined target sequences

  • In vitro translation systems supplemented with the recombinant protein

• Expression systems comparison: Different expression systems have been used successfully, as evidenced by the commercial availability of recombinant Nelumbo lutea rps7 from both E. coli and Baculovirus sources.

  Expression System	Advantages	Disadvantages
  E. coli	High yield, simple, cost-effective	May lack proper folding, potential endotoxin contamination
  Baculovirus	Better folding, post-translational modifications	More complex, lower yield, higher cost
  Yeast	Eukaryotic system, scalable	Different codon bias, glycosylation patterns

For optimal expression strategy, researchers should consider the intended experimental applications and required protein characteristics.

How does Nelumbo lutea rps7 compare with homologous proteins in other plant species at the functional level?

Comparative studies of rps7 across plant species reveal both conservation and specialization:

The evolutionary conservation of rps7 structure and function across diverse plant species suggests its fundamental importance in chloroplast translation, while species-specific variations may reflect adaptations to different ecological niches or photosynthetic requirements.

What insights does the Nelumbo lutea genome provide about chloroplast ribosomal protein evolution compared to other basal eudicots?

The Nelumbo lutea genome provides valuable insights into chloroplast ribosomal protein evolution:

Understanding the evolution of chloroplast ribosomal proteins in Nelumbo provides a window into the adaptation of the photosynthetic machinery across plant evolution and may inform studies of chloroplast function in other species.

How can recombinant Nelumbo lutea rps7 be used to study chloroplast translation regulation in vitro?

Recombinant Nelumbo lutea rps7 offers several approaches to study chloroplast translation regulation:

• Reconstituted translation systems: Purified recombinant rps7 can be incorporated into in vitro chloroplast translation systems to:

  • Assess its role in translation initiation, elongation, and termination

  • Study how alterations in rps7 concentration affect translation rates of different chloroplast mRNAs

  • Investigate potential regulatory interactions with translation factors

• RNA binding studies: Given that S7 proteins can bind to specific RNA motifs, researchers can:

  • Perform systematic evolution of ligands by exponential enrichment (SELEX) to identify high-affinity binding motifs

  • Use electrophoretic mobility shift assays (EMSAs) to quantify binding to candidate regulatory RNA sequences

  • Apply RNA immunoprecipitation followed by sequencing (RIP-seq) with anti-rps7 antibodies to identify target transcripts in chloroplast extracts

• Structure-function analyses: Based on findings from bacterial systems that show S7 uses the same determinants to bind rRNA and mRNA , researchers can:

  • Generate point mutations in key residues (N-terminal region, loop 2, loop 5)

  • Test how these mutations affect binding to different RNA targets

  • Correlate structural changes with functional outcomes in translation assays

• Regulatory circuit reconstruction: The autoregulatory function of S7 in bacteria (binding to its own mRNA to repress translation) suggests similar mechanisms may operate in chloroplasts. In vitro approaches can:

  • Test if Nelumbo lutea rps7 binds preferentially to its own mRNA

  • Determine the concentration dependence of this binding

  • Assess how environmental factors affect this regulatory circuit

These approaches can help elucidate the complex interplay between ribosome assembly and translational regulation in chloroplasts, which remains less understood than the equivalent processes in bacteria.

What role might rps7 play in chloroplast stress responses, and how can this be experimentally investigated?

The potential role of rps7 in chloroplast stress responses and experimental approaches to investigate it:

• Conceptual framework:

  • Chloroplast translation is known to respond to various stresses (light, temperature, oxidative)

  • Ribosomal proteins with dual functions as translational regulators could serve as stress response mediators

  • S7's ability to bind both rRNA and mRNA positions it as a potential regulatory factor

• Evidence from related research:

  • Studies have shown that Nelumbo species have adapted to various ecological stresses over millions of years

  • Recombinant chloroplast proteins can maintain activity under varied conditions, suggesting potential roles in stress adaptation

  • In bacteria, ribosomal protein overexpression (including S7) can cause growth defects, indicating their regulation is critical

• Experimental approaches:

  Approach	Methodology	Expected Outcome
  Transcriptomics	RNA-seq of Nelumbo under various stresses (heat, cold, drought, high light)	Changes in rps7 expression patterns relative to other chloroplast genes
  Proteomics	Quantitative mass spectrometry of chloroplast ribosomes under stress conditions	Altered stoichiometry or post-translational modifications of rps7
  In vitro translation	Chloroplast translation assays with varying rps7 concentrations under stress-mimicking conditions	Differential translation of specific transcripts
  RNA interactome	RNA immunoprecipitation of rps7 followed by sequencing under normal and stress conditions	Stress-specific changes in the RNA targets bound by rps7
  Structural studies	Cryo-EM of chloroplast ribosomes under different conditions	Conformational changes in rps7 and its interactions

• Specific stress connections to investigate:

  • Thermogenesis: American lotus flowers are known to be thermogenic (heat-generating) , suggesting specialized metabolic adaptations that may involve unique chloroplast functions

  • Aquatic adaptations: As an aquatic plant, Nelumbo lutea faces unique stresses related to water chemistry, submerged conditions, and light filtration

  • Oxidative stress: The anti-inflammatory properties of Nelumbo extracts suggest robust antioxidant systems that may involve chloroplast regulation

Understanding how rps7 contributes to stress responses could provide insights into the mechanisms of environmental adaptation in lotus and potentially inform strategies for improving stress tolerance in crops.

What are the promising applications of CRISPR/Cas9 genome editing for studying rps7 function in Nelumbo lutea?

CRISPR/Cas9 genome editing offers several promising applications for studying rps7 function in Nelumbo lutea:

• Precise gene modification approaches:

  • Point mutations: Create specific amino acid substitutions in conserved regions of rps7 to assess their impact on chloroplast translation and plant phenotype

  • Domain swapping: Replace domains of Nelumbo lutea rps7 with those from other species (e.g., Nelumbo nucifera, bacterial homologs) to investigate functional conservation

  • Regulatory element editing: Modify the promoter or untranslated regions of rps7 to alter expression patterns and study regulatory networks

• Technical considerations for chloroplast genome editing:

  • Unlike nuclear genome editing, chloroplast transformation typically relies on homologous recombination

  • CRISPR/Cas9 systems specifically designed for chloroplast genome editing are being developed

  • Delivery methods must account for the multiple copies of the chloroplast genome present in each cell

• Experimental designs and expected outcomes:

  Editing Approach	Methodology	Expected Outcome	Research Value
  Knockout/knockdown	Disrupt rps7 coding sequence	Potentially lethal or severe growth defects	Essential nature of rps7 for chloroplast function
  Reporter fusion	Tag rps7 with fluorescent protein	Visualization of protein localization and dynamics	Insights into regulatory mechanisms
  Inducible expression	Create conditionally regulated rps7 variants	Controlled modulation of rps7 levels	Effects of rps7 abundance on translation
  Mutant libraries	Generate series of mutations across rps7	Range of phenotypes based on mutation severity	Structure-function relationships

• Integration with other techniques:

  • Combine genome editing with transcriptomics and proteomics to assess global impacts

  • Use edited lines for biochemical studies of altered rps7 function

  • Apply edited material in ecological studies to assess fitness effects

How might structural biology approaches advance our understanding of Nelumbo lutea rps7 function in the chloroplast ribosome?

Advanced structural biology approaches offer significant potential for understanding Nelumbo lutea rps7 function:

• Cryo-electron microscopy (cryo-EM):

  • Recent advances have enabled high-resolution structures of complete chloroplast ribosomes (e.g., spinach chloroplast ribosome at 3.4Å resolution)

  • Application to Nelumbo lutea chloroplast ribosomes could reveal:

    • Specific interactions between rps7 and 16S rRNA

    • Contacts with other ribosomal proteins

    • Conformational changes during translation

    • Species-specific features compared to other plant chloroplast ribosomes

• X-ray crystallography of isolated components:

  • Crystallization of recombinant Nelumbo lutea rps7 alone or in complex with RNA targets

  • Co-crystallization with interacting proteins or translation factors

  • Structural studies of rps7 mutations to understand functional domains

• NMR spectroscopy for dynamics:

  • Study solution dynamics of rps7-RNA interactions

  • Investigate conformational changes upon binding to different RNA targets

  • Examine interactions with regulatory factors

• Integrative structural biology approaches:

  • Combine multiple techniques (cryo-EM, crystallography, NMR, mass spectrometry)

  • Use computational modeling to predict interactions and dynamic properties

  • Apply hydrogen/deuterium exchange mass spectrometry to map interaction surfaces

• Specific research questions addressable through structural biology:

  • How does the structure of Nelumbo lutea rps7 compare to bacterial and other plant rps7 proteins?

  • What structural features enable rps7 to recognize both rRNA and mRNA targets?

  • How do post-translational modifications affect rps7 structure and function?

  • Are there conformational changes in rps7 under different physiological conditions?

• Technical considerations:

  • Purification of intact chloroplast ribosomes from Nelumbo lutea requires specialized protocols

  • Sample heterogeneity can complicate structural determination

  • Integrating structural data with functional assays is essential for meaningful interpretation