Recombinant Thermus thermophilus 50S ribosomal protein L17 (rplQ)

What is Thermus thermophilus 50S ribosomal protein L17 and its functional significance?

Thermus thermophilus L17 (encoded by the rplQ gene) is one of the core proteins of the large (50S) ribosomal subunit in this extreme thermophile. It plays a structural role in ribosome assembly and integrity, particularly at the high temperatures (65-75°C) at which T. thermophilus thrives. L17 has been identified as one of the proteins retained in functionally active subribosomal particles, suggesting its importance for peptidyl transferase activity . Within the ribosomal architecture, L17 contributes to maintaining the tertiary structure of the ribosome and participates in the formation of the peptidyl transferase center.

How can recombinant T. thermophilus L17 be cloned and expressed in E. coli?

The recombinant expression of T. thermophilus L17 can be achieved using the following protocol:

• Gene synthesis and codon optimization: The synthetic gene of T. thermophilus rplQ should be codon-optimized for expression in E. coli without altering the amino acid sequence .

• Vector construction: The gene can be cloned into an expression vector such as pET21b or pET26b with an N-terminal His6-tag and a TEV protease recognition site for purification purposes .

• Transformation and expression:

  • Transform the construct into E. coli BL21(DE3) cells

  • Grow transformed cells to OD600 of 0.6 at 37°C in LB medium with appropriate antibiotic

  • Induce protein expression with 0.5 mM IPTG

  • Allow expression to continue for 5 hours at 28°C

• Cell harvest and lysis:

  • Collect cells and resuspend in buffer (50 mM Tris-HCl pH 8, 1.5 M KCl, 2 mM MgCl2, 1 mM TCEP)

  • Use a cell cracker or sonication for lysis

  • Separate supernatant by centrifugation at 25,000 g for 30 minutes

What purification methods are effective for recombinant T. thermophilus L17?

The thermostability of T. thermophilus proteins enables unique purification strategies:

• Heat treatment: Exploit the thermostability of T. thermophilus L17 to perform a heat precipitation step (70-80°C for 20 minutes) to denature most E. coli proteins while leaving the target protein intact .

• Affinity chromatography: Purify His-tagged L17 using Ni-NTA chromatography with the following buffer conditions:

  • Binding buffer: 50 mM Tris-HCl pH 8, 500 mM NaCl, 20 mM imidazole

  • Washing buffer: 50 mM Tris-HCl pH 8, 500 mM NaCl, 50 mM imidazole

  • Elution buffer: 50 mM Tris-HCl pH 8, 500 mM NaCl, 300 mM imidazole

• Tag removal: When necessary, use TEV protease digestion to remove the His-tag.

• Size exclusion chromatography: Perform a final purification step using size exclusion chromatography with a buffer containing 50 mM Tris-HCl pH 8, 150 mM NaCl.

Typical yields of recombinant L17 from T. thermophilus can reach 30-35 mg/L of E. coli culture, comparable to yields reported for other thermostable proteins like RecA .

What is the structural position of L17 in the T. thermophilus ribosome?

L17 occupies a strategic position in the 50S ribosomal subunit of T. thermophilus. Based on cryo-EM and crystallographic studies:

How does L17 contribute to ribosome assembly and thermostability?

L17 plays critical roles in ribosome assembly and thermostability through several mechanisms:

• Assembly pathway: L17 is one of the early-binding proteins in the 50S subunit assembly pathway, creating a scaffold for subsequent protein and rRNA interactions.

• Thermostability mechanisms:

  • Contains a higher proportion of charged and hydrophobic residues compared to mesophilic homologs

  • Forms salt bridges that are particularly stable at high temperatures

  • Exhibits increased hydrogen bonding with rRNA

  • Contains fewer thermolabile residues (Asn, Gln, Met, Cys)

• RNA-protein interactions: L17 forms multiple contacts with 23S rRNA domains, stabilizing their tertiary structure. This interaction is crucial for maintaining the functional conformation of the ribosome at elevated temperatures.

• Post-translational modifications: Unlike some other ribosomal proteins, L17 does not appear to undergo extensive methylation or other modifications that might contribute to thermostability .

How can genetic manipulation of rplQ in T. thermophilus be achieved?

Genetic manipulation of the rplQ gene in T. thermophilus can be accomplished through several approaches:

• Natural competence-based transformation: T. thermophilus exhibits natural competence, allowing direct uptake of DNA . This can be utilized to introduce targeted mutations in the rplQ gene.

• Gene replacement strategies:

  • Create a construct containing modified rplQ flanked by homologous regions

  • Include a selectable marker (e.g., kanamycin resistance gene kat/htk)

  • Transform into T. thermophilus and select for recombinants

• Counter-selection systems: Use the p-chlorophenylalanine (p-Cl-Phe) counter-selection system for the sequential introduction of unmarked mutations :

  • The p-Cl-Phe system allows selection of cells that have undergone a second recombination event to remove the selectable marker

  • This creates "clean" mutations without antibiotic resistance markers

• Phenotypic screening: Screen transformants for temperature-sensitive phenotypes, which may indicate successful modification of rplQ, similar to observations in other ribosomal protein mutants .

What techniques can be used to study L17 interactions within the ribosome?

Several advanced techniques can be employed to investigate L17 interactions within the ribosome:

• Cryo-electron microscopy (cryo-EM):

  • Has been successfully used to visualize T. thermophilus ribosomal structures at resolutions of 3-7 Å

  • Can reveal the positioning of L17 and its interactions with rRNA and other proteins

  • Particularly useful for comparative studies between active and hibernating ribosomes

• Cross-linking coupled with mass spectrometry (XL-MS):

  • Can identify specific interaction sites between L17 and other ribosomal components

  • UV or chemical cross-linkers can be used to capture transient interactions

  • MS analysis identifies the cross-linked residues

• MALDI-TOF mass spectrometry:

  • Useful for analyzing post-translational modifications of L17

  • Can be used to determine the exact mass of L17 and identify any modifications

• Ribosome profiling:

  • Provides insights into how L17 mutations might affect translation dynamics

  • Can reveal ribosome pausing or altered elongation rates

What is the role of L17 in ribosome hibernation and stress response?

L17 participates in the formation of hibernating 100S ribosomes in T. thermophilus, which are inactive ribosome dimers formed in response to cellular stresses . The specific role of L17 in this process includes:

• Structural stabilization: L17 maintains its position in the 100S ribosome, contributing to the stability of the hibernating complex.

• Interaction with hibernation factors: The hibernation-promoting factor (TtHPF) in T. thermophilus interacts with the ribosome to facilitate dimerization. While direct interaction with L17 has not been explicitly demonstrated, the positioning of L17 near the interface region suggests potential involvement.

• Stress response regulation: The exact ratio of hibernation factors to ribosomes (1:1) is critical for optimal dimerization . L17 may contribute to the recognition of these factors during stress conditions.

• Species-specific dimerization: Unlike in E. coli, T. thermophilus ribosome dimerization does not involve direct ribosome-ribosome interactions . This unique feature may involve L17 in stabilizing the specific conformation required for dimerization via hibernation factors.

How does T. thermophilus L17 compare to homologs in other bacterial species?

T. thermophilus L17 exhibits both conservation and adaptation compared to homologs in other bacteria:

• Sequence conservation:

  • Core functional domains are conserved across bacterial species

  • Higher conservation in rRNA binding regions

  • Thermophilic adaptations in non-critical regions

• Thermophilic adaptations:

  • Increased proportion of charged residues (Arg, Lys, Glu)

  • Decreased content of thermolabile residues (Asn, Gln)

  • Enhanced hydrophobic core stabilization

• Genetic context conservation:

  • The gene order rpl36-rps13-rps11-rps4-rpoA-rpl17 is conserved across diverse bacterial species

  • This suggests functional importance of this arrangement, possibly for coordinated expression

• Functional role:

  • Consistently found in active subribosomal particles across species

  • One of the eight proteins (along with L2, L3, L13, L15, L18, L21, and L22) retained in minimal active particles

Can T. thermophilus L17 be used as a model for studying ribosomal protein evolution in extremophiles?

T. thermophilus L17 serves as an excellent model for studying ribosomal protein evolution in extremophiles for several reasons:

• Thermophilic adaptations: The protein exhibits characteristic adaptations that enhance stability at high temperatures, providing insights into evolutionary mechanisms for thermal adaptation.

• Structural conservation: Despite adaptations for thermostability, L17 maintains its core structural features and ribosomal positioning, allowing for meaningful comparative analyses.

• Experimental advantages:

  • The thermostability of T. thermophilus L17 facilitates structural studies

  • Simplified purification protocols due to heat stability

  • Natural competence of T. thermophilus enables genetic manipulation

• Evolutionary perspectives:

  • Comparisons between mesophilic and thermophilic L17 proteins reveal selection pressures and adaptive strategies

  • Conservation of the genetic context suggests evolutionary constraints on ribosomal protein gene organization

  • Potential insights into the co-evolution of ribosomal proteins and rRNA

What are common issues in recombinant T. thermophilus L17 expression and how can they be addressed?

Researchers may encounter several challenges when working with recombinant T. thermophilus L17:

• Inclusion body formation:

  • Problem: High expression levels may lead to aggregation and inclusion body formation

  • Solution: Lower the expression temperature to 20°C, reduce IPTG concentration to 0.1-0.2 mM, or use solubility-enhancing fusion tags (SUMO, MBP)

• Incomplete translation:

  • Problem: Despite codon optimization, incomplete translation products may occur

  • Solution: Use E. coli strains with additional tRNAs for rare codons (Rosetta, CodonPlus)

• Purification challenges:

  • Problem: Co-purification of E. coli ribosomal components

  • Solution: Include additional washing steps with high salt (1M NaCl) and low concentrations of imidazole (50 mM)

• Activity assessment:

  • Problem: Difficulty in measuring individual L17 activity

  • Solution: Evaluate incorporation into partial ribosomal assembly assays or use binding assays with labeled 23S rRNA fragments

How can the interaction between L17 and rRNA be studied in vitro?

Several methods can be employed to study L17-rRNA interactions in vitro:

• Electrophoretic mobility shift assays (EMSA):

  • Purified L17 is incubated with labeled 23S rRNA fragments

  • Binding is visualized as shifted bands on native gels

  • Can determine binding affinity (Kd) through titration experiments

• Filter binding assays:

  • Rapid quantitative assessment of protein-RNA interactions

  • Can be performed at varying temperatures to assess thermostability of interactions

• Surface plasmon resonance (SPR):

  • Real-time binding kinetics measurements

  • Allows determination of kon and koff rates

  • Can be performed at different temperatures to simulate thermophilic conditions

• Reconstitution assays:

  • In vitro reconstitution of partial or complete ribosomal subunits

  • Can be performed with wild-type or mutant L17 to assess contribution to assembly

  • Sucrose gradient analysis to evaluate incorporation efficiency

• Structural techniques:

  • X-ray crystallography or cryo-EM of L17-rRNA complexes

  • Nuclear magnetic resonance (NMR) for studying dynamics of interactions

  • Chemical probing of rRNA structure in the presence/absence of L17

What potential applications exist for engineered T. thermophilus L17 variants?

Engineered T. thermophilus L17 variants hold promise for several applications:

• Enhanced ribosome engineering:

  • Creation of specialized ribosomes with altered translation properties

  • Development of ribosomes that can function in non-natural environments

  • Incorporation of non-canonical amino acids at enhanced efficiency

• Thermostable biosensors:

  • L17-based binding domains for RNA detection systems

  • Temperature-resistant diagnostic tools

  • Environmental monitoring applications

• Structural biology tools:

  • Thermostable crystallization chaperones

  • Stabilizing components for membrane protein studies

  • Fusion partners for challenging protein expression

• Antibiotic development:

  • Novel targets for antimicrobial development

  • Structures of thermophilic L17-antibiotic complexes may reveal new binding modes

  • Screening platforms for identifying compounds that disrupt ribosome assembly

How might CRISPR-Cas technologies enhance T. thermophilus L17 research?

CRISPR-Cas technologies offer new opportunities for T. thermophilus L17 research:

• Precise genome editing:

  • Introduction of point mutations in the native rplQ gene

  • Creation of tagged versions for tracking and purification

  • Generation of conditional knockdowns to study essentiality

• Transcriptional regulation:

  • CRISPRi systems adapted for thermophilic growth conditions

  • Tunable expression of L17 to study dosage effects

  • Simultaneous regulation of multiple ribosomal components

• In vivo tracking:

  • CRISPR-based imaging of L17 during ribosome assembly

  • Real-time monitoring of expression under stress conditions

  • Studies of co-localization with other ribosomal components

• High-throughput mutant libraries:

  • Saturation mutagenesis of L17 to identify critical residues

  • Selection for enhanced thermostability or altered function

  • Parallel assessment of fitness effects across growth conditions

• Adaptation for thermophilic conditions:

  • Engineering of Cas proteins for optimal activity at high temperatures

  • Development of guide RNAs with enhanced stability at elevated temperatures

  • Integration with existing natural competence systems in T. thermophilus