Recombinant Butomus umbellatus 30S ribosomal protein S7, chloroplastic (rps7)

What is the biological function of chloroplastic rps7 in Butomus umbellatus?

The S7 protein in the chloroplast of Butomus umbellatus functions as a structural component of the 30S small ribosomal subunit, playing a critical role in protein synthesis. Beyond its structural role, evidence suggests that the S7 protein binds to the 5′ untranslated regions (5′UTRs) of several chloroplast genes and may serve either a general or specific regulatory function in translation initiation within the chloroplast . Unlike its bacterial homolog in E. coli, which represses translation by sequestering the Shine-Dalgarno (SD) sequence, the chloroplastic S7 protein appears to activate rather than repress translation initiation . This regulatory role may be particularly significant in Butomus umbellatus, which is adapted to various aquatic environments.

How is the rps7 gene organized in the Butomus umbellatus genome?

The rps7 gene is present in both the chloroplast and mitochondrial genomes of Butomus umbellatus. In the chloroplast, it is located in the inverted repeat regions of the plastome, making it a double-copy gene along with other genes such as rpl2, rpl23, rps12, ndhB, and ycf2 . In the mitochondrial genome, rps7 is one of only four ribosomal protein genes (along with rps1, rps3, and rps12) retained in Butomus umbellatus . This dual presence in both organellar genomes is noteworthy, as many ribosomal protein genes have been lost from plant mitochondrial genomes during evolution. Additionally, in the S. suchowensis mitochondrial genome, rps7 has been found to have an intact copy within a large repeat region (R1) .

What RNA editing patterns are observed in rps7 transcripts?

RNA editing is a post-transcriptional process that converts specific cytidines to uridines in organellar genomes of land plants. While the specific RNA editing sites in Butomus umbellatus rps7 have not been directly reported in the provided materials, RNA editing analysis in related species suggests that rps7 likely undergoes some degree of editing. In S. suchowensis, for example, 330 RNA editing sites were predicted across 33 protein-coding genes, with 36.1% occurring at the first base position of codons and 63.9% at the second position . These edits often convert amino acids from hydrophilic to hydrophobic (46.7%), potentially affecting protein structure and function. Experimental determination of RNA editing sites in Butomus umbellatus rps7 would require:

• RT-PCR amplification of rps7 transcripts

• cDNA sequencing and comparison with the genomic sequence

• Identification of C-to-U conversion sites

What methodologies are recommended for expressing and purifying recombinant Butomus umbellatus rps7 protein?

Based on successful approaches with other ribosomal proteins, the following methodology is recommended for expressing and purifying recombinant Butomus umbellatus rps7:

Expression System Protocol:

• Gene Amplification and Cloning:

  • Amplify the rps7 coding region from Butomus umbellatus chloroplast DNA using PCR with primers targeting the 5'UTR and N-terminal region (forward) and 3'UTR and C-terminal region (reverse)

  • Include appropriate restriction sites (e.g., BamHI and EcoRI) in primers for directional cloning

  • Clone the amplified sequence into an expression vector such as pGEX-4T-1 for expression as a GST fusion protein

• Transformation and Expression:

  • Transform the construct into E. coli BL21 or similar expression strain

  • Induce protein expression with IPTG (0.5-1.0 mM) at 18-25°C for 4-6 hours to minimize inclusion body formation

  • Verify expression by SDS-PAGE analysis

• Protein Purification:

  • Lyse cells using a French press or sonication in appropriate buffer (e.g., PBS with protease inhibitors)

  • Purify the fusion protein using glutathione-Sepharose affinity chromatography

  • Cleave the GST tag using thrombin (if using pGEX-4T-1 vector)

  • Further purify using size exclusion chromatography if necessary

• Quality Control:

  • Verify protein purity by SDS-PAGE and Western blotting

  • Confirm protein identity by mass spectrometry

  • Assess protein folding by circular dichroism spectroscopy

How can researchers study the RNA-binding specificity of recombinant rps7?

To investigate the RNA-binding properties of recombinant Butomus umbellatus rps7 protein, researchers can employ several complementary approaches:

Experimental Approaches for RNA-Binding Studies:

• Electrophoretic Mobility Shift Assays (EMSA):

  • Generate in vitro transcribed RNA probes corresponding to potential binding targets (e.g., 5'UTRs of various chloroplast genes)

  • Incubate purified recombinant rps7 protein with labeled RNA probes

  • Analyze binding by native gel electrophoresis to detect mobility shifts

  • Include competition assays with unlabeled RNA to assess specificity

• UV Crosslinking and RNA Immunoprecipitation:

  • Incubate recombinant rps7 with target RNAs and apply UV crosslinking

  • Immunoprecipitate the protein-RNA complexes using anti-rps7 antibodies

  • Analyze the bound RNAs by RT-PCR or sequencing

• Surface Plasmon Resonance (SPR):

  • Immobilize either the protein or RNA on a sensor chip

  • Measure real-time binding kinetics and determine association and dissociation constants

  • Test RNA constructs with mutations in predicted binding sites to map the interaction interface

• RNA Structural Analysis:

  • Perform RNA structure probing (e.g., SHAPE, RNase mapping) of target RNAs with and without bound rps7

  • Identify structural changes induced by protein binding

  • Generate secondary structure models of binding sites

What approaches can be used to investigate the role of rps7 in chloroplast translation regulation?

The regulatory role of rps7 in chloroplast translation can be investigated using multiple complementary approaches:

Methods for Studying Translation Regulation:

• In vitro Translation Assays:

  • Develop a chloroplast-specific in vitro translation system using isolated chloroplast ribosomes

  • Test the effect of adding or depleting recombinant rps7 on translation efficiency

  • Use reporter constructs with different 5'UTRs to assess sequence-specific effects

• Ribosome Profiling:

  • Isolate chloroplast ribosomes from Butomus umbellatus under different conditions

  • Sequence ribosome-protected mRNA fragments

  • Analyze ribosome occupancy and translation efficiency across the chloroplast transcriptome

  • Compare results between wild-type and rps7-depleted systems

• Cryo-EM Structural Analysis:

  • Purify chloroplast ribosomes with bound rps7

  • Perform cryo-EM to determine the structural features of the rps7-ribosome complex

  • Focus on the architecture of the 30S small subunit to understand how rps7 contributes to ribosome function

  • Compare with existing structures of chloroplast ribosomes, such as the 3.7 Å resolution structure of spinach chloroplast 30S small subunit

• Genetic Manipulation:

  • Develop transformation protocols for Butomus umbellatus chloroplasts

  • Create mutants with altered rps7 expression or binding capacity

  • Assess the effects on chloroplast translation and plant phenotype

How do diploid and triploid cytotypes of Butomus umbellatus differ in gene expression patterns, including rps7?

Butomus umbellatus exhibits both diploid and triploid cytotypes in its global range, with interesting differences in their biology and reproductive strategies. While specific information on rps7 expression differences between cytotypes is not directly provided in the search results, several approaches can be used to investigate this question:

Methodological Approach for Cytotype Comparison:

• Transcriptome Analysis:

  • Perform RNA-seq on chloroplasts isolated from both diploid and triploid plants grown under identical conditions

  • Compare expression levels of chloroplast genes, including rps7

  • Analyze differential expression patterns across developmental stages and environmental conditions

• Proteomics Analysis:

  • Use quantitative proteomics to compare chloroplast protein abundance between cytotypes

  • Specifically quantify rps7 protein levels using targeted approaches such as selected reaction monitoring (SRM)

  • Analyze post-translational modifications that may differ between cytotypes

• Physiological Response Experiments:

  • Grow diploid and triploid plants under different nutrient conditions (e.g., varying N and P levels) as described in the research on Butomus umbellatus

  • Measure chloroplast function and protein synthesis rates

  • Correlate with rps7 expression and protein abundance

Research with other species has shown that triploid Butomus umbellatus plants generally have lower plasticity in response to environmental variations compared to diploid plants . Diploid plants produced 172% more reproductive biomass and 57% more total biomass across levels of nitrogen, and 158% more reproductive biomass and 33% more total biomass across phosphorus levels than triploid plants . Such physiological differences may correlate with differential expression or function of chloroplast genes, potentially including rps7.

What is the current understanding of the structural features of rps7 that enable its RNA binding capabilities?

Understanding the structural basis of rps7's RNA binding function requires detailed structural analysis:

Structural Analysis Approaches:

• Homology Modeling:

  • Generate structural models of Butomus umbellatus rps7 based on available structures of S7 proteins from other species

  • Use cryo-EM structures of chloroplast ribosomes, such as the 3.7 Å resolution structure of spinach chloroplast 30S small subunit

  • Identify conserved RNA-binding motifs and predict their interactions with RNA targets

• X-ray Crystallography:

  • Crystallize purified recombinant rps7 protein alone and in complex with target RNA sequences

  • Determine high-resolution structures to identify precise binding interfaces

  • Compare with bacterial S7 structures to understand functional divergence

• NMR Spectroscopy:

  • Use solution NMR to study the dynamics of rps7-RNA interactions

  • Map RNA binding sites through chemical shift perturbation experiments

  • Investigate conformational changes upon RNA binding

• Site-Directed Mutagenesis:

  • Create point mutations in predicted RNA-binding residues

  • Assess effects on binding affinity and specificity using the techniques described in FAQ #6

  • Correlate structural features with functional outcomes

The structure of chloroplast ribosomal proteins, including rps7, reveals unique localization that provides mechanistic insights into chloroplastic translation . The chloroplast ribosome has acquired plastid-specific ribosomal proteins (PSRPs) during evolution, which appear to play important regulatory roles in translation .

What methods are recommended for studying the interaction between rps7 and other components of the chloroplast translation machinery?

To investigate how rps7 interacts with other components of the chloroplast translation machinery:

Interaction Analysis Methods:

• Co-Immunoprecipitation (Co-IP):

  • Generate antibodies specific to Butomus umbellatus rps7

  • Perform Co-IP experiments using chloroplast extracts

  • Identify interacting proteins by mass spectrometry

  • Confirm interactions through reciprocal Co-IP experiments

• Yeast Two-Hybrid and Split-Ubiquitin Assays:

  • Screen for protein-protein interactions between rps7 and other translation-related proteins

  • Verify positive interactions with alternative methods

  • Map interaction domains through truncation analysis

• Bimolecular Fluorescence Complementation (BiFC):

  • Create fusion constructs of rps7 and potential interacting partners with split fluorescent proteins

  • Transform into appropriate plant expression systems

  • Visualize interactions through fluorescence microscopy in vivo

• In vitro Reconstitution Experiments:

  • Purify recombinant components of the chloroplast translation machinery

  • Assemble partial or complete complexes in vitro

  • Study the role of rps7 in complex assembly and function

  • Use techniques such as gradient centrifugation, light scattering, or analytical ultracentrifugation to monitor complex formation