Recombinant Thermus thermophilus 30S ribosomal protein S13 (rpsM)

What makes Thermus thermophilus suitable as a model organism for ribosomal protein research?

T. thermophilus is an extremely thermophilic bacterium found globally in high-temperature environments, belonging to a phylum that branches deep within the Bacteria domain alongside Deinococcus and other genera . It offers several advantages as an experimental system:

• Aerobic growth at an optimum temperature of 65°C allows for straightforward laboratory cultivation

• Natural competence for transformation with either chromosomal or plasmid DNA

• One of the most efficient and rapid DNA uptake machinery yet described

• Several developed genetic tools including shuttle vectors, host integration systems, and gene expression reporter systems

• Thermostable proteins that maintain structural integrity under conditions that would denature mesophilic proteins

These characteristics make T. thermophilus particularly valuable for structural and functional studies of ribosomal components, including the S13 protein.

What structural characteristics define the T. thermophilus 30S ribosomal protein S13?

The 30S ribosomal protein S13 from T. thermophilus possesses several key structural features:

• High content of alpha-helical secondary structure as revealed by preliminary NMR studies

• A distinctive C-terminal domain (CTD) tail that is longer than its counterpart in E. coli

• Strategic positioning within the ribosome structure, with the CTD tail located close to and interacting with A-site and P-site tRNAs

This structural arrangement suggests important functional roles in the translation process, particularly in relation to tRNA positioning and movement during protein synthesis.

How does T. thermophilus S13 differ from S13 proteins in mesophilic bacteria?

The most notable difference between T. thermophilus S13 and mesophilic counterparts (such as E. coli S13) is the length and positioning of the C-terminal domain (CTD) tail:

• T. thermophilus possesses a longer CTD tail compared to E. coli

• The T. thermophilus S13 CTD tail is positioned closer to and interacts more extensively with A-site and P-site tRNAs in ribosome structures

• This structural difference may represent an adaptation to high-temperature environments and contribute to ribosome stability under extreme conditions

These variations in structure likely contribute to differences in translation dynamics between thermophilic and mesophilic organisms, with potential implications for protein synthesis rates and accuracy.

What are the optimal growth conditions for maintaining T. thermophilus cultures?

Based on established protocols, T. thermophilus requires specific growth conditions:

These conditions support robust growth while maintaining the thermophilic characteristics of the organism .

What transformation methods are most effective for genetic manipulation of T. thermophilus?

T. thermophilus transformation can be performed following the method described by Koyama et al. :

• Prepare recipient T. thermophilus cells (strain HB27 is commonly used)

• Transform with plasmid or genomic DNA using the Koyama protocol

• Select transformants on TEM plates containing appropriate selection agents:

  • 30 μg/ml kanamycin sulfate for kanamycin resistance markers

  • 15 mM p-Cl-Phe (p-chlorophenylalanine) for alternative selection

• Incubate plates at 65°C until colonies appear

The natural competence of T. thermophilus facilitates higher transformation efficiency compared to many other bacterial species, making genetic manipulation relatively straightforward .

What selection markers and reporter systems are available for T. thermophilus?

Several genetic tools have been developed for T. thermophilus:

Selection markers:

• Antibiotic resistance genes evolved to function at high temperatures:

  • kat and htk encoding kanamycin adenyltransferases

  • hph encoding hygromycin B phosphotransferase

  • ble gene for additional selection

• Chemical selection using 15 mM p-chlorophenylalanine (p-Cl-Phe)

Reporter systems:

• β-galactosidase system based on thermostable variants

• β-glucosidase system

• System based on the crtB gene encoding phytoene synthase (involved in carotenoid biosynthesis)

These tools enable sophisticated genetic engineering approaches for studying ribosomal proteins and other gene functions in T. thermophilus.

What expression systems are suitable for producing recombinant T. thermophilus S13?

The expression of T. thermophilus S13 has been successfully achieved in E. coli expression systems :

• Cloning approach:

  • PCR amplification of the rpsM gene from T. thermophilus genomic DNA

  • Cloning into appropriate E. coli expression vectors

  • Sequence verification of the cloned gene

• Expression conditions:

  • Standard E. coli expression systems can be used

  • Temperature, inducer concentration, and duration may require optimization

  • Lower induction temperatures may improve folding despite the thermostable nature of the protein

• Host considerations:

  • E. coli BL21(DE3) or derivatives are commonly used for heterologous protein expression

  • Specialized strains for expression of thermophilic proteins may provide advantages in certain cases

What structural analysis techniques are most informative for studying S13?

Several complementary techniques provide valuable structural information about S13:

• NMR spectroscopy:

  • Preliminary NMR studies have revealed a high content of alpha-helical secondary structure in T. thermophilus S13

  • Solution-state NMR can provide detailed information about protein dynamics, particularly in flexible regions like the CTD tail

• X-ray crystallography:

  • While not explicitly mentioned in the search results for isolated S13, crystallography of complete ribosomes has revealed S13's position and interactions

• Complementary approaches:

  • Circular dichroism (CD) spectroscopy for secondary structure analysis

  • Thermal denaturation studies to assess stability

  • Molecular dynamics simulations to model flexibility and interactions

These approaches collectively provide insights into the structural basis of S13 function within the ribosome.

What is the role of the C-terminal tail of S13 in ribosomal function?

The C-terminal domain (CTD) tail of S13 plays a critical role in translation, as evidenced by experimental manipulations of its length:

• Structural positioning:

  • The CTD tail is positioned between the A-site tRNA and P-site tRNA in ribosome structures

  • This strategic location suggests involvement in tRNA movement or positioning during translation

• Species-specific variations:

  • The length of the CTD tail differs between species

  • T. thermophilus has a longer CTD tail than E. coli

  • The longer tail in T. thermophilus shows more extensive interaction with A-site and P-site tRNAs

• Functional impact:

  • Modification of the CTD tail length affects translocation rates during protein synthesis

  • The tail appears to influence multiple steps in the translation process, including potentially ribosome recycling, tRNA selection accuracy, and peptide release

How do modifications to the S13 CTD tail affect ribosomal function?

Experimental studies using modified S13 proteins have revealed significant functional consequences:

These findings indicate that:

• The extended CTD tail significantly delays translocation, likely by physically interfering with tRNA movement

• Both shortening and extending the tail cause growth defects, suggesting optimal length is important

• The similar growth defects but different translocation impacts between modifications suggest multiple stages of translation may be affected

What experimental approaches can measure the impact of S13 modifications on translation?

Several experimental approaches provide quantitative assessment of S13's role in translation:

• In vitro translation assays:

  • Measurement of tripeptide formation times using purified ribosomes

  • Quantification of translation rates with different S13 variants

  • Analysis of specific translation steps (initiation, elongation, termination)

• In vivo functional assessment:

  • Growth rate measurements under various conditions

  • Polysome profile analysis to assess ribosome assembly and activity

  • Protein synthesis rate determination using pulse-labeling techniques

• Structural analysis of ribosomes with modified S13:

  • Cryo-EM studies to visualize conformational changes

  • Structure-function correlations between S13 modifications and ribosomal activity

  • Monitoring of ribosome dynamics during the translation cycle

These approaches collectively provide a comprehensive understanding of how S13 modifications affect the translation process.

How should experiments investigating S13 function be designed?

When designing experiments to study S13 function, researchers should follow systematic experimental design principles:

• Define research variables:

  • Independent variables: S13 sequence variations, experimental conditions

  • Dependent variables: Growth rates, translation rates, structural parameters

  • Control for extraneous variables such as temperature, media composition, and strain background

• Formulate testable hypotheses:

  • Null hypothesis: "S13 modifications do not affect translation parameters"

  • Alternative hypothesis: "Specific S13 modifications affect translation in predictable ways"

• Design treatments with appropriate controls:

  • Include wild-type S13 as baseline control

  • Consider both truncation and extension modifications

  • Include site-specific mutations to test function of specific residues

• Randomize and replicate:

  • Use randomized block design to account for batch effects

  • Include sufficient biological and technical replicates

  • Calculate appropriate sample sizes for statistical power

• Employ multiple measurement approaches:

  • Combine in vitro and in vivo assessments

  • Use complementary structural and functional analyses

  • Document all experimental parameters thoroughly

What are the key considerations when engineering S13 variants for functional studies?

When creating S13 variants for functional studies, several considerations are important:

• Modification strategies:

  • λ-red recombineering for chromosomal modifications

  • Site-directed mutagenesis for specific amino acid changes

  • Domain swapping between species (e.g., T. thermophilus and E. coli)

• Target regions:

  • C-terminal domain tail (shown to affect translocation)

  • Interfaces with other ribosomal components

  • Residues conserved across species or unique to thermophiles

• Expression considerations:

  • Ensure similar expression levels between variants

  • Verify proper incorporation into ribosomes

  • Check for potential effects on ribosome assembly

• Control constructs:

  • Include conservative substitutions that maintain charge and size

  • Create variants with known phenotypes as positive controls

  • Introduce mutations in non-conserved regions as negative controls

How can researchers leverage T. thermophilus as a platform for biotechnology applications?

T. thermophilus offers unique advantages as a biotechnology platform, particularly for applications requiring thermostable components:

• High-temperature biocatalysis:

  • Production of engineered enzymes in T. thermophilus for reactions at elevated temperatures

  • Development of whole-cell biocatalysts for high-value compound synthesis

  • Utilization of T. thermophilus ribosomes and translation machinery for in vitro protein synthesis

• Environmental advantages:

  • Reactions that tolerate hotter ambient temperatures

  • Reduced need for expensive cooling systems

  • Future-proof approach to biocatalysis in the context of increasing global temperatures

• Methodological approaches:

  • Recombinant protein expression in T. thermophilus

  • Engineering of thermostable ribosomes with modified S13 and other components

  • Development of high-temperature fermentation processes

• Potential applications:

  • Production of medicines and materials under high-temperature conditions

  • Development of thermostable diagnostic reagents

  • Creation of heat-resistant biocatalysts for industrial processes

These approaches leverage the natural thermophilic properties of T. thermophilus while applying modern molecular biology techniques to develop practical biotechnology applications.