Recombinant Idiomarina loihiensis 30S ribosomal protein S13 (rpsM)
Description
Introduction
Idiomarina loihiensis 30S ribosomal protein S13 (rpsM) is a component of the 30S ribosomal subunit in Idiomarina loihiensis, a marine bacterium . Ribosomal proteins are essential for protein synthesis, and S13 plays a crucial role in the assembly and function of the ribosome .
Characteristics of Ribosomal Protein S13
Ribosomal protein S13 is a highly conserved protein found in both prokaryotic and eukaryotic organisms . In Escherichia coli, S13 is located in the head of the 30S subunit and is part of the S7 assembly branch .
2.1. Primary Structure
Rat S13 contains 150 amino acids and has a molecular weight of 17,080 . The mRNA for the protein is about 620 nucleotides in length .
2.2. Assembly and Function
S13 is essential for the assembly and function of the 30S ribosomal subunit . It interacts with 16S rRNA and other ribosomal proteins to form a stable and functional subunit .
Idiomarina loihiensis
Idiomarina loihiensis is a Gram-negative, halophilic bacterium isolated from deep-sea hydrothermal vent fields . It belongs to the Idiomarina genus and is known for its ability to thrive in extreme environments .
Recombinant Production
Recombinant Idiomarina loihiensis 30S ribosomal protein S13 (rpsM) can be produced in various expression systems, such as E. coli or yeast . The recombinant protein is often used for structural and functional studies .
4.1. Applications of Recombinant S13
Recombinant S13 is used in studies to understand its role in ribosome assembly, protein synthesis, and antibiotic resistance . It can also be used to investigate its interactions with other ribosomal proteins and RNA .
Role in Drug Resistance and Apoptosis
Ribosomal protein S13 has been implicated in multi-drug resistance in gastric cancer cells by suppressing drug-induced apoptosis . Overproduction of rpS13 in mammalian cells interferes with splicing of its own pre-mRNA by a feedback mechanism .
Regulation of Expression
Human ribosomal protein S13 regulates the expression of its own gene through a feedback mechanism . S13 inhibits the excision of intron 1 from rpS13 pre-mRNA .
6.1. Autoregulation
Autoregulation is a mechanism by which the level of each individual ribosomal protein in the cell could be independently controlled . This may be crucial for the extraribosomal functions of ribosomal proteins .
Interactions with Other Molecules
S13 interacts with other members of the S7 assembly branch . It binds specifically to a transcript containing intron 1 and flanking exon sequences .
Relevant data
Product Specs
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Target Background
KEGG: ilo:IL1894
STRING: 283942.IL1894
Q&A
What is the genomic context of the rpsM gene in Idiomarina loihiensis?
The rpsM gene encoding the 30S ribosomal protein S13 is part of the complete genome of Idiomarina loihiensis, a deep-sea γ-proteobacterium with a single circular chromosome of 2,839,318 base pairs with 47% GC content. The genome contains 2,640 predicted open reading frames (ORFs), four rRNA operons (16S-23S-5S), and 56 tRNA genes, accounting for 92.1% of the genome . Ribosomal proteins, including S13, are essential components of this organism's translational machinery, which has evolved unique adaptations to the extreme conditions of deep-sea hydrothermal vent environments.
How does S13 contribute to the structure of the 30S ribosomal subunit in I. loihiensis?
S13 is positioned in the head of the 30S ribosomal subunit, which is one of the three distinct structural domains (head, body, and platform) that compose the mature 30S subunit. Each structural domain corresponds to a portion of 16S rRNA and a subset of ribosomal proteins . Based on studies in other bacteria, S13 is part of the 3' major domain family of proteins and plays a crucial role in maintaining the structural integrity of the head region. Its position is over 100 Å away from proteins like S20, which reside near the bottom of the body of the 30S subunit . This spatial organization is critical for proper ribosomal function and assembly.
What is the assembly pathway for S13 in the 30S ribosomal subunit?
Research on ribosomal assembly patterns suggests that S13 in I. loihiensis, similar to its E. coli counterpart, is likely part of the S7 assembly branch. This means that S13 depends on the prior association of S7 with the 16S rRNA for its incorporation into the assembling 30S subunit . This dependency pathway differs from earlier models that incorrectly positioned S13 as dependent on S20. The correct positioning of S13 in the assembly map is consistent with structural data showing the spatial separation between S13 and S20 in the mature ribosome .
Why is I. loihiensis S13 of interest for extremophile adaptation studies?
I. loihiensis was isolated from hydrothermal vents on the Lō`ihi Seamount, Hawaii, and survives in a wide range of growth temperatures (4°C to 46°C) and salinities (0.5% to 20% NaCl) . The ribosomal proteins, including S13, must function under these extreme conditions, making them valuable models for studying protein adaptations to extreme environments. Unlike obligate anaerobic vent hyperthermophiles, I. loihiensis inhabits partially oxygenated cold waters at the periphery of hydrothermal vents, representing a different adaptive strategy .
How do evolutionary adaptations in I. loihiensis S13 differ from other Idiomarina species in varying environments?
Comparative genomic analyses of Idiomarina species reveal that despite being isolated from geographically and geologically similar environments, different Idiomarina strains often show higher relatedness to other Idiomarina species than to each other . This suggests that geographic isolation has contributed to population divergence within the Idiomarina genus. For S13 specifically, its sequence conservation is likely balanced with adaptive modifications that maintain core functionality while accommodating the specific physiochemical constraints of different extreme environments.
What experimental approaches are optimal for expressing recombinant I. loihiensis S13?
Table 1: Recommended Expression Systems for Recombinant I. loihiensis S13
| Expression System | Advantages | Limitations | Optimization Strategy |
|---|---|---|---|
| E. coli BL21(DE3) | High yield, simple protocol | Potential misfolding due to different environmental conditions | Use cold-shock induction (16°C), optimize codon usage |
| E. coli Arctic Express | Better folding at lower temperatures | Lower yield | Extended expression time (24-48h) |
| Cell-free system | Avoids toxicity issues, rapid | Higher cost, lower scalability | Supplement with chaperones and correct ion concentrations |
Based on structural studies of ribosomal proteins, a recommended approach involves:
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Gene synthesis with codon optimization for E. coli
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Cloning into a vector with a removable His-tag
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Expression in E. coli Arctic Express at 16°C for 24h
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Purification under native conditions with IMAC followed by gel filtration
This approach addresses the challenge of expressing proteins from extremophiles in mesophilic hosts while maintaining structural integrity.
How can researchers investigate the role of S13 in I. loihiensis ribosome assembly in vitro?
To investigate S13's role in ribosome assembly, researchers can employ base-specific chemical footprinting and primer extension analysis similar to methods used for E. coli S13 . The recommended experimental design would include:
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Isolation of 16S rRNA from I. loihiensis
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In vitro reconstitution experiments with combinations of purified recombinant ribosomal proteins
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Addition or omission of S13 to determine dependency relationships
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Chemical modification of the rRNA-protein complex with reagents like dimethyl sulfate
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Primer extension analysis to map the binding sites protected by S13
These approaches can provide insights into whether I. loihiensis S13 follows similar assembly patterns to E. coli S13, particularly regarding its dependence on S7 rather than S20 .
What are the implications of S13's position in the ribosomal assembly map for in vivo studies?
The correct positioning of S13 in the S7 assembly branch rather than as dependent on S20 has significant implications for in vivo studies. Research on E. coli has demonstrated that ribosomal assembly in vivo can show remarkable plasticity and redundancy not observed in vitro . For I. loihiensis S13, this suggests:
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Knockout studies might not yield expected phenotypes if compensatory mechanisms exist
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Assembly chaperones specific to extreme environments may facilitate S13 incorporation
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Temperature and salt concentration might significantly affect assembly pathways involving S13
Experiments designed to test these hypotheses should account for the unique growth conditions of I. loihiensis (wide temperature and salinity ranges) and consider the possible discrepancies between in vitro and in vivo assembly mechanisms.
How can researchers purify functional I. loihiensis S13 while maintaining its native conformation?
Table 2: Purification Protocol Optimization for I. loihiensis S13
| Step | Procedure | Critical Parameters | Quality Control |
|---|---|---|---|
| Lysis | Sonication in high-salt buffer | 50 mM Tris pH 7.5, 500 mM NaCl, 5 mM MgCl₂ | Clear lysate, no precipitation |
| IMAC | Ni-NTA chromatography | 10-250 mM imidazole gradient | >90% purity by SDS-PAGE |
| Tag removal | TEV protease digestion | 1:50 ratio, 4°C overnight | Complete cleavage by mass spec |
| Polishing | Size exclusion chromatography | Superdex 75, 25 mM Tris pH 7.5, 300 mM NaCl | Single peak, >95% purity |
| Functional test | 16S rRNA binding assay | 37°C, 1:1 molar ratio | Gel shift assay positive |
The key challenge in purifying functional S13 from I. loihiensis is maintaining its conformation while removing it from its native ribosomal context. The high-salt buffers throughout purification mimic the native high-salt environment of this marine extremophile , helping to maintain proper folding and solubility.
What analytical techniques best characterize the interaction between I. loihiensis S13 and 16S rRNA?
Multiple complementary techniques should be employed to thoroughly characterize the S13-16S rRNA interaction:
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Isothermal Titration Calorimetry (ITC): Measures binding thermodynamics and stoichiometry, particularly valuable for comparing wild-type and mutant S13 proteins. Similar approaches have been used successfully for other ribosomal proteins like the S6/S18 heterodimer .
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Cryo-Electron Microscopy: Provides structural information on S13's position within the assembled ribosome at near-atomic resolution, allowing comparison with structures from other species.
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Chemical Footprinting: Identifies the specific nucleotides in 16S rRNA that are protected by S13 binding, revealing the molecular details of the interaction interface .
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Surface Plasmon Resonance: Determines association and dissociation rates at different salt concentrations and temperatures, mimicking the range of conditions I. loihiensis experiences in its natural habitat.
How should researchers design experiments to investigate the impact of extreme conditions on S13 function?
When investigating S13 function under extreme conditions relevant to I. loihiensis' habitat, experiments should:
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Test protein stability and rRNA binding across the full temperature range (4-46°C) and salinity range (0.5-20% NaCl) that I. loihiensis tolerates .
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Use circular dichroism spectroscopy to monitor secondary structure changes at different temperatures and salt concentrations.
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Employ in vitro translation assays with S13-depleted and S13-reconstituted ribosomes under varying conditions to measure functional impact.
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Compare the behavior of I. loihiensis S13 with homologs from non-extremophilic organisms to identify adaptations specific to extreme environments.
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Use mutagenesis to identify residues critical for thermostability and halotolerance.
This systematic approach will help delineate how S13 contributes to I. loihiensis' remarkable adaptability to extreme conditions.
How can researchers reconcile discrepancies between in vitro and in vivo ribosomal assembly data for S13?
Studies on E. coli have shown that in vivo ribosomal assembly exhibits greater plasticity than predicted by in vitro studies . To reconcile potential discrepancies for I. loihiensis S13:
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Complementary approaches: Combine in vitro reconstitution experiments with in vivo studies using fluorescently tagged S13 to track its incorporation into ribosomes.
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Quantitative proteomics: Compare stoichiometry of ribosomal proteins in native ribosomes versus in vitro reconstituted particles.
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Assembly intermediates: Isolate and characterize assembly intermediates from cells grown under different conditions to identify alternative assembly pathways.
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Computational modeling: Develop models that incorporate flexibility in assembly pathways and predict the impact of environmental conditions on assembly order.
The goal should be to develop a unified model that explains both in vitro dependencies and in vivo plasticity, similar to how the S15 dependency pattern has been clarified through multiple approaches .
What comparative genomic approaches can reveal functional adaptations in I. loihiensis S13?
Table 3: Comparative Genomics Framework for S13 Functional Analysis
| Analysis Type | Data Required | Expected Outcomes | Interpretation Approach |
|---|---|---|---|
| Sequence conservation | S13 sequences from multiple Idiomarina species | Identification of conserved and variable regions | Map to structure, identify pressure-related adaptations |
| Codon usage analysis | rpsM gene sequences, whole genome codon usage | Translation efficiency patterns | Correlate with expression levels under different conditions |
| Synteny analysis | Genomic context of rpsM across species | Operonic structure, co-regulated genes | Identify regulatory differences in extremophiles |
| Positive selection analysis | S13 sequences from diverse environments | Sites under positive selection pressure | Correlate with functional domains and interfaces |
This comparative framework can reveal how I. loihiensis S13 has evolved specific adaptations to function in extreme environments. For example, the high percentage of iso-branched fatty acids in Idiomarina suggests membrane adaptations to extreme conditions, and S13 may have co-evolved features that optimize ribosome-membrane interactions in this context.
How should researchers interpret functional data from recombinant S13 in heterologous systems?
When interpreting functional data for recombinant I. loihiensis S13 expressed in heterologous systems:
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Context dependency: Consider that S13 normally functions within a complex network of RNA-protein and protein-protein interactions that may not be fully recapitulated in heterologous systems.
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Environmental factors: Account for differences between standard laboratory conditions and I. loihiensis' native extremophile conditions when interpreting activity and stability.
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Post-translational modifications: Verify whether any modifications present in native S13 are missing in the recombinant protein and assess their functional importance.
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Comparison controls: Always include S13 proteins from model organisms as controls to benchmark functional parameters.
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Validation strategies: Confirm key findings using complementary approaches, such as in vitro reconstitution followed by in vivo complementation tests.
This cautious approach to interpretation acknowledges the challenges of studying proteins from extremophiles in standard laboratory systems.
What are common issues in expressing recombinant I. loihiensis S13 and how can they be resolved?
Table 4: Troubleshooting Guide for I. loihiensis S13 Expression
| Problem | Possible Causes | Solutions |
|---|---|---|
| Low expression yield | Codon bias, toxicity to host | Use codon-optimized gene, tight expression control, specialized host strains |
| Inclusion body formation | Improper folding, high expression rate | Lower induction temperature (16°C), use solubility tags (SUMO, MBP) |
| Proteolytic degradation | Recognition by host proteases | Include protease inhibitors, use protease-deficient strains |
| Poor binding to 16S rRNA | Misfolding, missing co-factors | Include Mg²⁺ in buffers, co-express with chaperones |
| Aggregation during purification | Hydrophobic interactions, salt-dependent stability | Optimize salt concentration based on I. loihiensis' natural environment |
Each troubleshooting strategy addresses a specific challenge in working with proteins from extremophiles in standard laboratory systems, with particular attention to the unique environmental adaptations of I. loihiensis.
How can researchers address contradictory results when comparing S13 assembly patterns between different studies?
To address contradictory results in S13 assembly pattern studies:
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Standardize experimental conditions: Carefully control temperature, salt concentration, and Mg²⁺ levels, which significantly impact ribosomal assembly.
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Verify protein quality: Confirm that recombinant S13 is properly folded using circular dichroism and thermal shift assays before assembly experiments.
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Assess RNA integrity: Check the integrity and proper folding of 16S rRNA used in reconstitution experiments.
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Investigate kinetic factors: Consider that assembly pathways may differ kinetically rather than thermodynamically, and time-resolved experiments may reconcile apparent contradictions.
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Evaluate methodology differences: Compare chemical footprinting, cryo-EM, and biochemical approaches to identify method-specific artifacts.
As demonstrated with E. coli S13, which was initially thought to depend on S20 but later correctly positioned in the S7 assembly branch , careful methodological analysis can resolve apparent contradictions in ribosomal assembly pathways.
What novel approaches could advance our understanding of I. loihiensis S13 function in extremophile adaptation?
Several innovative approaches show promise for deepening our understanding of S13's role in extremophile adaptation:
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High-pressure structural biology: Study S13 structure and interactions under pressure conditions mimicking deep-sea environments to understand adaptations specific to I. loihiensis' habitat.
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In-cell NMR: Monitor S13 dynamics within living I. loihiensis cells under various environmental stresses to capture physiologically relevant conformational changes.
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Ancestral sequence reconstruction: Resurrect ancestral S13 sequences to trace the evolutionary path leading to extremophile adaptations.
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Ribosome profiling: Compare translation dynamics in ribosomes with native versus mutant S13 to identify functional consequences of adaptive mutations.
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Single-molecule FRET: Directly observe S13's role in ribosomal dynamics during translation under varying environmental conditions.
These approaches can reveal how S13 contributes to I. loihiensis' remarkable adaptability to deep-sea hydrothermal vent environments.
How might synthetic biology approaches utilizing I. loihiensis S13 advance extremophile research?
Synthetic biology applications incorporating I. loihiensis S13 could include:
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Engineering ribosomes with enhanced function under extreme conditions by incorporating S13 adaptations into mesophilic organisms.
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Creating chimeric ribosomes to study which domains of S13 are critical for extremophile adaptation.
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Developing biosensors based on S13 stability under various conditions to monitor environmental parameters.
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Using I. loihiensis S13 as a model to design stabilized proteins for industrial applications requiring extreme temperature or salt tolerance.
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Exploring minimal ribosome designs that incorporate essential features from extremophile S13 to create synthetic translation systems with expanded environmental operating ranges.
These applications could not only advance fundamental understanding of ribosome adaptation but also yield biotechnological innovations for extreme environments.
