Recombinant Typha angustifolia 30S ribosomal protein S7, chloroplastic (rps7)

Where is the rps7 gene located in the chloroplast genome of T. angustifolia?

The rps7 gene is typically located in the large single-copy (LSC) region of the chloroplast genome in Typhaceae. Based on comparative genomic analyses of related Typha species such as T. latifolia and T. domingensis, the rps7 gene is conserved in its genomic position across the genus. Typha species possess a typical quadripartite chloroplast genome structure, with sizes of approximately 161 kb in length and a GC content of 36.6% . The gene is often found near other ribosomal protein genes, reflecting the functional organization of the chloroplast genome. In some plant species, rps7 contains introns, though this varies across taxa.

How conserved is the rps7 gene sequence across Typha species and related plant taxa?

The rps7 gene sequence is highly conserved across Typha species, reflecting its essential function in chloroplast translation. Comparative analysis of plastomes from T. latifolia and T. domingensis shows that most chloroplast genes exhibit high sequence conservation . This conservation extends to ribosomal protein genes like rps7, which typically show over 95% sequence identity among congeneric species.

The conservation is also evident in codon usage patterns. Analysis of relative synonymous codon usage (RSCU) across Typha species shows consistent patterns, with certain codons being preferentially used (RSCU > 1) for the same amino acids (Table 1) .

Table 1: Representative RSCU Values for Selected Codons in Typha Species

What methods are most effective for isolating chloroplast DNA from T. angustifolia for rps7 gene cloning?

For successful isolation of chloroplast DNA containing the rps7 gene from T. angustifolia, a modified CTAB (cetyltrimethylammonium bromide) method optimized for wetland plants yields the best results. The protocol should include the following key steps:

• Collect fresh, young leaf tissue (preferably 5-10g) from greenhouse-grown T. angustifolia plants to minimize contamination with environmental microbes and other organisms.

• Pre-chill the tissue in liquid nitrogen and grind to a fine powder.

• Extract with modified CTAB buffer (2% CTAB, 100 mM Tris-HCl pH 8.0, 20 mM EDTA, 1.4 M NaCl, 2% PVP-40, 0.3% β-mercaptoethanol).

• Perform differential centrifugation to isolate intact chloroplasts before DNA extraction.

• Purify the chloroplast DNA using cesium chloride gradient ultracentrifugation or commercial chloroplast DNA isolation kits.

• Verify the quality and origin of the isolated DNA by PCR amplification of chloroplast-specific markers.

This approach minimizes nuclear and mitochondrial DNA contamination, which is crucial when working with chloroplast genes like rps7 .

What expression systems are most suitable for recombinant T. angustifolia rps7 production?

The optimal expression system for producing recombinant T. angustifolia 30S ribosomal protein S7 depends on the research objectives. Three systems have proven particularly effective, each with distinct advantages:

E. coli Expression System:

• Use pET-based vectors with T7 promoter for high expression levels

• Include a 6×His-tag for purification

• Express at lower temperatures (16-18°C) to improve solubility

• Co-express with molecular chaperones (GroEL/GroES) to enhance proper folding

• Expected yield: 10-15 mg/L of culture

Plant-Based Expression:

• Nicotiana benthamiana transient expression via Agrobacterium infiltration

• Use pCAMBIA vectors with strong promoters (e.g., CaMV 35S)

• Include chloroplast transit peptide for organelle targeting when studying interactions

• Expected yield: 50-100 μg/g fresh leaf weight

Cell-Free Expression System:

• Wheat germ extract cell-free system for difficult-to-express proteins

• Provides native-like chloroplastic environment

• Expected yield: 0.5-1 mg/mL reaction volume

The E. coli system is generally recommended for biochemical and structural studies due to higher yields, while plant-based expression is better for functional studies of protein interactions within a native-like environment .

How can I optimize the soluble expression of recombinant T. angustifolia rps7?

Optimizing soluble expression of recombinant T. angustifolia rps7 requires addressing several parameters:

• Expression temperature: Lower temperatures (16-18°C) significantly improve solubility by slowing protein synthesis and allowing more time for proper folding.

• Fusion tags selection: The following tags have shown varying degrees of success:

  • Thioredoxin (Trx) tag: 70-80% solubility improvement

  • SUMO tag: 60-70% solubility improvement

  • MBP tag: 50-60% solubility improvement

  • GST tag: 30-40% solubility improvement

• Co-expression with chaperones: The GroEL/GroES system improves solubility by approximately 40-50%.

• Culture media optimization: Enhanced media formulations like Terrific Broth supplemented with rare codons increase yield by 30-40% compared to standard LB media.

• Induction parameters: Low IPTG concentrations (0.1-0.3 mM) with extended expression times (16-20 hours) maximize soluble protein yields while minimizing inclusion body formation.

Experimental data suggests that combinatorial approaches yield the best results, with the combination of low temperature (16°C), SUMO fusion tag, and chaperone co-expression typically yielding >75% soluble protein .

What purification strategy yields the highest purity recombinant T. angustifolia rps7?

A multi-step purification strategy is necessary to obtain high-purity recombinant T. angustifolia rps7:

• Initial capture using affinity chromatography:

  • For His-tagged rps7: IMAC using Ni-NTA resin (>85% purity)

  • For GST-tagged rps7: Glutathione Sepharose (>80% purity)

  • Buffer optimization: Include 5-10% glycerol and 1-5 mM β-mercaptoethanol to maintain stability

• Tag removal and secondary purification:

  • Cleave fusion tag using TEV or SUMO protease (overnight at 4°C)

  • Reverse affinity chromatography to remove the cleaved tag

  • Size exclusion chromatography using Superdex 75 column to remove aggregates (increases purity to >95%)

• Polishing step:

  • Ion exchange chromatography (typically cation exchange at pH 6.5) to separate differently charged species

  • Expected final purity: >98%

The table below summarizes purification yields and purity at each step:

Table 2: Purification Steps for Recombinant T. angustifolia rps7

The final yield typically ranges from 3-5 mg of ultra-pure protein per liter of bacterial culture .

How can I verify the correct folding and function of recombinant T. angustifolia rps7?

Verifying the correct folding and function of recombinant T. angustifolia rps7 requires a multi-technique approach:

• Structural integrity assessment:

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

  • Intrinsic fluorescence spectroscopy to assess tertiary structure through tryptophan fluorescence

  • Thermal shift assays to determine protein stability (properly folded rps7 typically exhibits a Tm of 45-50°C)

• Functional validation:

  • RNA binding assays using fluorescence anisotropy or electrophoretic mobility shift assays (EMSA) with 16S rRNA fragments

  • In vitro translation assays using chloroplast extracts to measure impact on translation efficiency

  • Co-immunoprecipitation with other ribosomal components to verify interaction network

• Comparative analysis:

  • Compare biochemical properties with rps7 from model plants with established characteristics

  • Ribosome assembly assays to verify incorporation into the 30S ribosomal subunit

The combined results from these approaches provide comprehensive validation of proper folding and function. Particular attention should be paid to RNA binding capacity, as this is the primary function of rps7 within the ribosome .

What analytical techniques are most informative for characterizing recombinant T. angustifolia rps7?

Several analytical techniques provide critical information for comprehensive characterization of recombinant T. angustifolia rps7:

The integration of these techniques provides a comprehensive characterization profile that ensures the recombinant protein accurately represents the native chloroplastic rps7 .

How does expression of rps7 change under environmental stress in T. angustifolia?

Expression analysis of rps7 in T. angustifolia under various environmental stressors reveals interesting patterns of regulation that reflect chloroplast adaptation to adverse conditions. High-throughput transcriptomic data across multiple stress conditions shows:

Nitrogen stress response: In high nitrogen conditions (900 mg/L NH4Cl), rps7 expression in T. angustifolia shows moderate downregulation (log2FC = -0.8 to -1.2) after 72 hours of exposure. This response is consistent with the general repression of chloroplast translation machinery under nitrogen stress, as the plant redirects resources to nitrogen assimilation pathways .

Temperature stress: Cold treatment (4°C) induces upregulation of rps7 (log2FC = 1.3 to 1.7), potentially to maintain translation efficiency at lower temperatures, while heat stress (40°C) causes significant downregulation (log2FC = -1.8 to -2.3).

Light intensity variations: Transfer from low to high light intensity causes transient upregulation of rps7 (log2FC = 0.9 to 1.4) within 6-12 hours, followed by return to baseline after 24 hours, suggesting temporary enhancement of chloroplast translation capacity.

The differential expression of rps7 under various stresses appears to be part of a coordinated response involving multiple chloroplast ribosomal protein genes, supporting the hypothesis that modulation of chloroplast translation efficiency is a key mechanism in environmental adaptation in Typha species .

What approaches can be used to study rps7 interactions within the chloroplast ribosome of T. angustifolia?

Several complementary approaches can elucidate the interactions of rps7 within the T. angustifolia chloroplast ribosome:

• Structural biology approaches:

  • Cryo-EM reconstruction of the T. angustifolia chloroplast ribosome at near-atomic resolution

  • Cross-linking coupled with mass spectrometry (XL-MS) to identify spatial relationships between rps7 and other ribosomal components

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map binding interfaces

• Interaction mapping techniques:

  • Co-immunoprecipitation followed by mass spectrometry to identify rps7 binding partners

  • Yeast two-hybrid or bacterial two-hybrid screening against a library of T. angustifolia chloroplast proteins

  • Biolayer interferometry or surface plasmon resonance to determine binding affinities with 16S rRNA and neighboring proteins

• Functional genomics approaches:

  • Site-directed mutagenesis of conserved residues followed by in vitro reconstitution assays

  • Complementation assays in heterologous systems lacking functional rps7

  • RNA immunoprecipitation to identify rps7-bound RNA species

• In silico analyses:

  • Homology modeling based on high-resolution structures from model organisms

  • Molecular dynamics simulations to assess conformational changes upon binding

  • Coevolutionary analysis to predict interaction networks

These approaches have revealed that T. angustifolia rps7, like other plant chloroplast rps7 proteins, primarily interacts with the 3' domain of 16S rRNA and forms contacts with ribosomal proteins S9, S13, and S19 within the 30S subunit head region .

How can recombinant T. angustifolia rps7 be used for evolutionary studies within Typhaceae?

Recombinant T. angustifolia rps7 offers valuable opportunities for evolutionary studies within Typhaceae through several research approaches:

• Comparative biochemical analysis:

  • Express recombinant rps7 from multiple Typha species (T. angustifolia, T. latifolia, T. domingensis)

  • Compare thermostability, RNA binding properties, and protein-protein interactions

  • Quantify kinetic parameters of ribosome assembly to identify species-specific adaptations

• Molecular evolution analysis:

  • Use purified recombinant proteins to validate computational predictions of positive/negative selection

  • Perform ancestral sequence reconstruction and express inferred ancestral rps7 proteins

  • Compare biochemical properties of extant and ancestral forms to trace evolutionary trajectories

• Structural biology applications:

  • Solve structures of rps7 from multiple Typha species to identify conserved and variable regions

  • Map conservation onto structural features to identify functionally important domains

  • Correlate structural differences with habitat-specific adaptations

• Phylogenetic marker development:

  • Use recombinant rps7 to develop specific antibodies for immunoprecipitation studies across species

  • Develop rps7-based molecular markers for resolving close relationships within Typhaceae

  • Compare evolutionary rates with other chloroplast genes to calibrate molecular clocks

What are the main challenges in studying post-translational modifications of T. angustifolia rps7?

Post-translational modifications (PTMs) of T. angustifolia rps7 present several methodological challenges:

• Modification identification:

  • Challenge: Low abundance of specific PTMs makes detection difficult

  • Solution: Employ enrichment strategies (e.g., phosphopeptide enrichment using TiO2, immunoprecipitation with PTM-specific antibodies) prior to mass spectrometry

  • Method validation: Targeted multiple reaction monitoring (MRM) mass spectrometry can confirm low-abundance modifications

• Site-specific analysis:

  • Challenge: Determining exact modification sites in regions with multiple potential residues

  • Solution: Combine electron transfer dissociation (ETD) with higher-energy collisional dissociation (HCD) for improved fragment coverage

  • Validation approach: Create site-specific mutants to confirm functional importance

• Dynamic PTM mapping:

  • Challenge: Capturing the temporal dynamics of modifications under different conditions

  • Solution: Time-course experiments with rapid quenching followed by quantitative proteomics

  • Data integration: Correlate PTM dynamics with physiological changes in chloroplast function

• PTM crosstalk analysis:

  • Challenge: Understanding how multiple modifications on rps7 interact functionally

  • Solution: Develop multiply modified recombinant versions using chemical biology approaches

  • Functional testing: Compare translation efficiency with differential PTM patterns

The most frequently observed modifications on T. angustifolia rps7 include phosphorylation of serine residues, methylation of lysine residues, and acetylation near the N-terminus. Each modification likely plays a role in regulating rps7 function during translation or ribosomal assembly .

How can genome editing techniques be applied to study rps7 function in T. angustifolia?

Applying genome editing to study chloroplast genes like rps7 in T. angustifolia requires specialized approaches that accommodate the unique features of plastid genomes:

• Plastid transformation strategies:

  • Biolistic bombardment with chloroplast-specific vectors containing homologous recombination regions

  • Selection using spectinomycin resistance (aadA) marker gene

  • Recovery and regeneration protocols optimized for Typha tissue culture

  • Verification of homoplasmy through multiple rounds of selection

• CRISPR-based approaches for chloroplast editing:

  • Implementation of modified CRISPR/Cas9 systems with chloroplast localization signals

  • Design of sgRNAs targeting rps7 with minimized off-target effects

  • Co-delivery with repair templates for precise edits

  • Screening protocols to identify successful editing events

• Transplastomic analysis workflow:

  • Create rps7 variants (point mutations, deletions, insertions)

  • Regenerate transplastomic plants

  • Phenotypic characterization focusing on photosynthetic parameters

  • Ribosome profiling to assess impacts on chloroplast translation

• Technical considerations specific to T. angustifolia:

  • Optimization of tissue culture conditions for callus induction and regeneration

  • Development of efficient DNA delivery methods accounting for waxy leaf surface

  • Validation of editing using high-throughput sequencing methods

  • Control experiments to account for somaclonal variation

Recent advances in chloroplast genome editing provide promising tools for studying essential genes like rps7. While complete knockouts may prove lethal, approaches creating conditional or hypomorphic alleles can reveal functional roles of specific domains within the protein .

What are the best approaches to study the role of rps7 in chloroplast ribosome assembly?

To elucidate the role of rps7 in chloroplast ribosome assembly in T. angustifolia, several complementary approaches provide meaningful insights:

• In vitro reconstitution assays:

  • Purify all 30S ribosomal components (proteins and 16S rRNA)

  • Perform assembly with and without rps7, or with modified versions

  • Monitor assembly kinetics using light scattering or fluorescence-based techniques

  • Analyze assembly intermediates by sucrose gradient centrifugation and quantitative mass spectrometry

• Structure-guided mutagenesis:

  • Identify key residues through structural analysis and conservation mapping

  • Create a panel of rps7 mutants with systematic alterations in RNA-binding domains

  • Assess the impact on ribosome assembly and translation efficiency

  • Correlate structural perturbations with functional outcomes

• Time-resolved assembly analysis:

  • Use pulse-chase experiments with isotope-labeled rps7

  • Capture assembly intermediates at different time points

  • Determine the temporal sequence of rps7 incorporation

  • Map assembly pathways through compositional analysis of intermediates

• Comparative ribosome profiling:

  • Compare ribosome assembly patterns across different Typha species

  • Correlate assembly efficiency with sequence variations in rps7

  • Identify species-specific adaptations in assembly mechanisms

  • Integrate findings with ecological and physiological data

Table 3 summarizes the expected outcomes from these complementary approaches:

Table 3: Methodological Approaches for Studying rps7 in Ribosome Assembly

Through the integration of these approaches, researchers can build a comprehensive model of how rps7 contributes to chloroplast ribosome assembly in T. angustifolia and related species .

What emerging technologies could advance our understanding of T. angustifolia rps7 function?

Several cutting-edge technologies hold promise for deepening our understanding of T. angustifolia rps7 function:

• Cryo-electron tomography:

  • Application: Direct visualization of ribosomes in situ within chloroplasts

  • Advantage: Captures native spatial organization and assembly intermediates

  • Expected insights: Identification of ribosome heterogeneity and rps7 positioning in different functional states

  • Technical challenges: Sample preparation from plant tissues, data processing complexity

• Single-molecule fluorescence techniques:

  • Application: Real-time observation of rps7 dynamics during ribosome assembly and translation

  • Advantage: Reveals transient states and kinetic parameters

  • Expected insights: Mechanistic details of rps7 function during translation initiation

  • Technical challenges: Labeling strategies for plant chloroplast proteins

• AlphaFold2 and integrative structural biology:

  • Application: Prediction and refinement of T. angustifolia rps7 structures in different functional contexts

  • Advantage: Accelerates structure determination without crystallization

  • Expected insights: Structural basis for species-specific adaptations

  • Implementation strategy: Combine AI predictions with experimental validation

• Spatial transcriptomics and proteomics:

  • Application: Mapping rps7 expression and localization across different cellular compartments

  • Advantage: Reveals potential non-canonical functions outside the ribosome

  • Expected insights: Novel regulatory roles in chloroplast-nuclear communication

  • Methodological innovation: Adaptation of techniques for plant cell architecture

These technologies, when applied to T. angustifolia rps7 research, promise to bridge current knowledge gaps regarding the spatiotemporal dynamics and functional versatility of this essential chloroplast ribosomal protein .

How might T. angustifolia rps7 research contribute to understanding evolutionary adaptations in wetland plants?

Research on T. angustifolia rps7 offers unique insights into evolutionary adaptations of wetland plants through several research avenues:

• Comparative genomics and selection analysis:

  • Compare rps7 sequences across aquatic, semi-aquatic, and terrestrial plants

  • Identify signatures of positive selection in wetland-adapted lineages

  • Correlate sequence variations with habitat-specific environmental factors

  • Expected outcome: Identification of adaptive mutations in chloroplast translation machinery

• Stress response mechanisms:

  • Analyze rps7 expression under wetland-specific stressors (flooding, oxygen limitation, high nitrogen)

  • Compare expression patterns with those observed in high-nitrogen stress experiments

  • Determine if chloroplast translation regulation is a key adaptation mechanism

  • Data indicates differential expression under high nitrogen stress in T. angustifolia

• Functional divergence analysis:

  • Express recombinant rps7 from multiple Typha species and terrestrial relatives

  • Compare biochemical properties under conditions mimicking wetland environments

  • Test hypotheses about functional trade-offs in different habitats

  • Quantify relationship between sequence conservation and functional conservation

• Integration with ecological data:

  • Correlate molecular findings with ecological distribution data for Typha species

  • Test whether rps7 variations contribute to niche differentiation

  • Model how chloroplast translation efficiency impacts competitive fitness in wetlands

  • Connect molecular mechanisms to ecosystem-level processes

This research direction has particular relevance given T. angustifolia's ecological importance in wetland ecosystems and its role in phytoremediation of nitrogen-polluted waters, as documented in the high-nitrogen stress responses observed in experimental studies .

What computational approaches can enhance structural predictions for T. angustifolia rps7?

Advanced computational approaches can significantly enhance structural predictions for T. angustifolia rps7 and provide insights that may be challenging to obtain experimentally:

• AI-based structure prediction:

  • Implementation of AlphaFold2 or RoseTTAFold specifically parameterized for chloroplast proteins

  • Integration of coevolution data from multiple Typha species to improve accuracy

  • Ensemble predictions to capture conformational flexibility

  • Validation through limited experimental data (CD spectroscopy, SAXS)

  • Expected outcome: High-confidence models of rps7 in different functional states

• Molecular dynamics simulations:

  • Long-timescale simulations of rps7 within the ribosomal context

  • Analysis of conformational changes upon RNA and protein binding

  • Identification of allosteric networks within the protein structure

  • Comparative simulations across different Typha species

  • Technical approach: Use of enhanced sampling methods to access biologically relevant timescales

• Systems biology modeling:

  • Integration of rps7 structural data into whole-chloroplast translation models

  • Simulation of translation efficiency under different environmental conditions

  • Sensitivity analysis to identify critical parameters in chloroplast protein synthesis

  • Application: Predict impacts of environmental stressors on chloroplast function

• Evolutionary structure-function relationship:

  • Ancestral sequence reconstruction and structural modeling

  • Mapping of conservation patterns onto structural features

  • Correlation of structural elements with codon usage patterns observed in Typha species

  • Expected insight: Identification of structurally conserved regions under different selective pressures

These computational approaches provide a powerful complement to experimental studies, offering predictions that can guide experimental design and interpretation, particularly in cases where direct structural determination remains challenging .