Recombinant Thermus thermophilus Translation initiation factor IF-2 (infB), partial

What is Translation Initiation Factor IF-2 in Thermus thermophilus and what is its role in protein synthesis?

Translation Initiation Factor IF-2 (infB) from Thermus thermophilus is a large GTP/GDP binding protein of approximately 63 kDa that plays a crucial role during the initiation phase of protein synthesis. It functions alongside two other essential initiation factors (IF1 and IF3) to ensure accurate placement of mRNA and tRNA in the 30S initiation complex .

IF2 specifically catalyzes the binding of initiator fMet-tRNA to the ribosomal P site in frame with mRNA, thereby increasing translation initiation rate and ensuring fidelity . The factor promotes the recruitment and stabilization of the initiator tRNA on the 30S initiation complex and facilitates the subsequent joining of the 50S ribosomal subunit to form the complete 70S initiation complex .

How does the structure of T. thermophilus IF2 compare to IF2 from mesophilic bacteria like E. coli?

T. thermophilus IF2 shows both similarities and differences when compared to its mesophilic counterpart from E. coli:

• Size difference: There is a substantial size difference between T. thermophilus IF2 (63 kDa) and E. coli IF2 (which is larger)

• Conserved domains: Despite size differences, there is reasonable structural conservation in key functional regions, particularly in the N-terminal ribosome binding region and the nucleotide binding domain

• Three-helix structure: The NMR structure for the N-terminus of the E. coli protein indicates that this three-helix structure is well conserved across species

• Functional compatibility: Despite structural variations, IF2 from both species are functionally interchangeable in in vitro translation systems, suggesting conservation of critical interaction surfaces

This functional compatibility is particularly remarkable given the extreme and highly divergent environments to which these species have adapted (T. thermophilus optimal growth at 72°C vs. E. coli at 37°C) .

What are the functional domains of T. thermophilus IF2 and what roles do they play?

T. thermophilus IF2 consists of several functional domains with specific roles in translation initiation:

When the N-terminal domain is deleted, proper positioning of fMet-tRNA and efficient transpeptidation are affected, demonstrating its critical role in the translation process .

What experimental techniques have been used to study the structure and function of T. thermophilus IF2?

Multiple complementary techniques have been employed to study T. thermophilus IF2:

• X-ray crystallography: Used to determine the 3D structure of IF2 components, with crystals diffracting to approximately 3.5 Å resolution

• Small Angle X-ray Scattering (SAXS): Revealed a more extended conformation of IF2 in solution than observed in crystal structures

• Cryo-electron microscopy (Cryo-EM): Visualized IF2 bound to ribosomes, providing insights into its conformation during translation initiation

• Fast kinetics and single-molecule fluorescence: Examined the dynamics of IF2-dependent ribosomal subunit joining and the role of the N-terminus

• Functional assays: GTP hydrolysis assays in ribosome-dependent manner to measure IF2 activity

• Coupled transcription-translation systems: Used to study IF2 function in protein synthesis and measure formation of full-length proteins

These diverse approaches collectively enable comprehensive structural and functional characterization of this important translation factor.

How can researchers optimize expression and purification of recombinant T. thermophilus IF2?

Optimizing expression and purification of recombinant T. thermophilus IF2 involves several key strategies:

Expression System:

• Expression in E. coli (e.g., BL21 strain) has been successful for T. thermophilus proteins

• IPTG induction (typically for 2 hours) has yielded good expression levels

Purification Strategy:

• Exploit thermostability: A single heat denaturation step of the E. coli S30 extract results in >90% purification, as E. coli proteins denature while T. thermophilus IF2 remains soluble

• Chromatography: Further purification can be achieved through column chromatography techniques

Sample Protein Purification Results:

Storage Considerations:

• Shelf life of liquid form: 6 months at -20°C/-80°C

• Shelf life of lyophilized form: 12 months at -20°C/-80°C

• Avoid repeated freeze-thaw cycles

• For short-term storage (up to one week), store working aliquots at 4°C

For reconstitution, it's recommended to use deionized sterile water at a concentration of 0.1-1.0 mg/mL with 5-50% glycerol as a cryoprotectant .

How does the thermostability of T. thermophilus IF2 compare to mesophilic homologs, and what structural features contribute to this thermostability?

T. thermophilus IF2 exhibits remarkable thermostability compared to mesophilic counterparts, aligned with the extreme thermophilic nature of the organism:

• Growth temperature comparison: T. thermophilus grows optimally at ~72°C with maximum growth at 80-83°C, while E. coli grows optimally at 37°C

• Thermal properties: The thermostability of T. thermophilus IF2 is evident in purification protocols, where a heat denaturation step (70°C) selectively denatures E. coli proteins while leaving T. thermophilus IF2 intact

While specific structural features contributing to IF2 thermostability haven't been fully characterized, general principles of thermophilic protein adaptation likely apply:

• Increased number of salt bridges and hydrogen bonds

• Enhanced hydrophobic core packing

• Reduced flexibility in loop regions

• Higher proportion of charged amino acids

• Shorter surface loops

These adaptations are consistent with broader studies of thermophilic proteins that show "thermozymes display higher stability and activity than their counterparts currently used in the biotechnological industry" .

What insights have been gained from cross-species compatibility studies of T. thermophilus IF2 with ribosomes from other bacteria?

Cross-species compatibility studies between T. thermophilus and E. coli translation components have revealed several important insights:

• Functional interchangeability: Translation initiation factors (IF1, IF2, IF3) from either species are equally effective in supporting protein synthesis in coupled transcription-translation systems

• Ribosomal compatibility: T. thermophilus ribosomes function with E. coli translational factors and tRNAs, despite their evolutionary distance

• Subunit interactions: T. thermophilus and E. coli ribosomal subunits can be combined to effect translation, with the spectrum of proteins produced depending upon the source of the 30S subunit

• Temperature effects: At 45°C (intermediate between optimal temperatures for both organisms), T. thermophilus ribosomes translate at ~25-30% of the maximal rate of E. coli ribosomes

This functional conservation suggests that "subunit-subunit interactions are highly conserved" and "each of the E. coli translational factors is capable of appropriate and functional interaction with T. thermophilus ribosomes" .

When examining factor combinations, experimental data shows:

• Addition of each factor individually did not stimulate translation

• The combination of IF1 and IF2 or IF1 and IF3 showed minimal activity

• The combination of IF2 and IF3 allowed translation to about half the level achieved with all three factors

• Maximal synthesis required all three initiation factors, regardless of source

What role does the N-terminal domain of T. thermophilus IF2 play in ribosomal subunit joining and translation efficiency?

• Stabilization of 70S complex: In the 70S initiation complex, the N-domain stabilizes interactions between IF2 and the L7/L12 stalk of the 50S ribosomal subunit

• tRNA positioning: When the N-domain is deleted, proper positioning of fMet-tRNA and efficient transpeptidation are affected

• Subunit joining dynamics: Fast kinetics and single-molecule fluorescence data demonstrate that the N-terminus promotes 70S initiation complex formation by stabilizing the productive sampling of the 50S subunit during 30S IC joining

• Structural transitions: The architecture of full-length IF2, determined by SAXS and cryo-EM, reveals a more extended conformation in solution and on the ribosome than observed in crystal structures, suggesting dynamic conformational changes facilitated by the N-domain

These findings highlight "the dynamics of IF2-dependent ribosomal subunit joining and the role played by the N terminus of IF2 in this process" .

What are the challenges in crystallizing full-length T. thermophilus IF2 and how can they be overcome?

Crystallizing full-length T. thermophilus IF2 presents several challenges that researchers have worked to overcome:

Primary Challenges:

• Large size (63 kDa) and domain flexibility

• Conformational heterogeneity during functional cycles

• Potential surface properties affecting crystal packing

Current Progress:
Initial crystallization trials of T. thermophilus IF2 have shown promising results, with at least one crystal diffracting to 3.5 Å resolution . The crystallization strategy employed involved:

• Ensuring high protein purity (demonstrated by SDS-PAGE analysis)

• Extensive crystallization trials

• Identification of ideal crystallization conditions

• Use of vapor diffusion technique with PEG/ion screening

Methodological Approaches:

• Construct optimization to address flexible regions

• Co-crystallization with binding partners to stabilize specific conformations

• High-throughput screening of crystallization conditions

• Alternative approaches such as cryo-EM for structural determination

The successful crystallization of T. thermophilus IF2, even at moderate resolution, represents significant progress in structural studies of this important translation factor.

How does T. thermophilus IF2 compare to other thermophilic bacterial translation factors?

Comparative studies of translation factors across thermophilic bacteria reveal both common features and species-specific adaptations:

• Conservation pattern: Like other translation factors from thermophiles, T. thermophilus IF2 shows higher conservation in functionally critical regions with more variation in peripheral regions

• Size variation: The size of IF2 varies across bacterial species. For example, Myxococcus xanthus IF2 is the largest known (1,070 residues) compared to the more compact T. thermophilus IF2 (63 kDa)

• Domain architecture: While core domains are conserved, thermophilic species show adaptations in domain structure and linking regions that may contribute to thermostability

• Functional temperature range: T. thermophilus translation factors function optimally at higher temperatures (70-80°C) compared to mesophilic counterparts, but can still function at lower temperatures (45°C) with reduced efficiency

Interestingly, not only IF2 but also IF1 and IF3 from thermophilic bacteria like Bacillus stearothermophilus are able to support translation with E. coli ribosomes, suggesting "a reasonably high level of conservation in all three IF interactions with the ribosome seems to be maintained" .

What role does T. thermophilus IF2 play in biotechnological applications?

T. thermophilus IF2, along with other thermostable proteins from this organism, offers significant biotechnological potential:

Advantages in Research Applications:

• Thermostability makes T. thermophilus proteins valuable for structural biology research

• T. thermophilus can be used as a host for selection and evolution of stable enzymes

• The thermostability simplifies purification protocols (using heat denaturation steps)

Potential Applications:

• In vitro translation systems: Creating thermostable translation systems capable of operating at elevated temperatures

• Structural biology: T. thermophilus components are preferred for crystallization and structural studies due to their stability

• Protein engineering: Understanding the thermostability determinants in T. thermophilus IF2 could inform the design of thermostable proteins for industrial applications

The T. thermophilus genome contains 2,218 putative genes, many with potential biotechnological interest . The recent completion of the genome sequencing project "will greatly improve our understanding" of fundamental cellular processes in thermophiles and expand the biotechnological toolkit .

How have genomic studies of T. thermophilus contributed to our understanding of translation factors in extreme thermophiles?

Genomic studies of T. thermophilus have significantly advanced our understanding of translation factors in extreme thermophiles:

• Genome characteristics: The T. thermophilus HB27 genome consists of a 1,894,877 base pair chromosome and a 232,605 base pair megaplasmid (pTT27)

• Gene identification: Genome analysis has enabled identification and characterization of all three translation initiation factors (IF1, IF2, IF3) in T. thermophilus

• Comparative genomics: Genome comparisons between T. thermophilus and mesophilic bacteria have highlighted adaptations in translation machinery for high-temperature environments

• Evolutionary insights: Genomic studies suggest that "thermal adaptation of protein synthesis in T. thermophilus was attributed to a key enzyme, a thiolase responsible for a post-transcriptional modification of the thermophilic bacterial tRNAs"

The genome sequence has also facilitated the development of genetic tools for T. thermophilus, including a recently reported CRISPR-Cas9 based system for genome editing that functions at 65°C . These advances enable more sophisticated genetic manipulation of T. thermophilus, allowing researchers to study translation factors through gene knockouts and modifications in their native context.

What strategies can be employed to study the GTPase activity of T. thermophilus IF2?

Studying the GTPase activity of T. thermophilus IF2 requires careful experimental design to account for its thermophilic nature:

Assay Methods:

• Ribosome-dependent GTP hydrolysis: IF2 can be assayed for its ability to hydrolyze GTP in a ribosome-dependent manner

• Spectrophotometric assays: Monitoring phosphate release using malachite green or other colorimetric methods

• Radiometric assays: Using [γ-32P]GTP to track hydrolysis rates

Reaction Conditions Optimization:

• Temperature considerations: Assays should be performed at elevated temperatures (45-70°C) to reflect the thermophilic nature of T. thermophilus IF2

• Buffer stability: Use thermostable buffers that maintain pH at high temperatures

• Divalent cation requirements: Ensure adequate Mg2+ or Mn2+ concentrations, as these are typically required for GTPase activity

Experimental Controls:

• Wild-type IF2 as positive control

• No-ribosome controls to determine background GTPase activity

• Heat-inactivated samples as negative controls

Data Analysis Parameters:

• Initial velocity measurements under steady-state conditions

• Determination of kinetic parameters (Km, kcat)

• Temperature dependence of activity (Arrhenius plots)

This methodological approach allows for detailed characterization of the GTPase activity that is central to IF2 function in translation initiation.

How can researchers investigate the interaction between T. thermophilus IF2 and fMet-tRNA?

Investigating the interaction between T. thermophilus IF2 and fMet-tRNA requires specialized techniques that account for the thermostable nature of the components:

Binding Assays:

• Filter binding assays: Using [35S]fMet-tRNAfMet to quantify binding to IF2

• Fluorescence techniques: Fluorescently labeled tRNA can be used to monitor binding through changes in anisotropy or FRET

• Surface plasmon resonance: Real-time measurement of association and dissociation kinetics

Experimental Setup:

• IF1 can produce an 8-fold increase in [35S]fMet-tRNAfMet binding to ribosomes in the presence of excess IF2

• Experiments should include controls with individual factors and factor combinations

Structural Approaches:

• Cryo-EM: Visualization of IF2-fMet-tRNA complexes on the ribosome

• Chemical crosslinking: Identification of contact points between IF2 and tRNA

• Hydroxyl radical footprinting: Mapping the interaction interface

Mutagenesis Studies:

• Site-directed mutagenesis of residues in the C-terminal domain of IF2 that are predicted to interact with fMet-tRNA

• Functional analysis of mutants to identify critical residues

These approaches can provide comprehensive insights into how T. thermophilus IF2 recognizes and positions the initiator tRNA during translation initiation.

What considerations are important when designing experiments to test the function of T. thermophilus IF2 in heterologous systems?

When designing experiments to test T. thermophilus IF2 function in heterologous systems (such as E. coli), several important considerations must be addressed:

Temperature Optimization:

• Experiments should be conducted at intermediate temperatures (e.g., 45°C) that balance the thermophilic requirements of T. thermophilus components with the tolerance of mesophilic systems

• Temperature gradients may be used to determine optimal conditions

Component Compatibility:

• When combining T. thermophilus IF2 with E. coli ribosomes, all three initiation factors (IF1, IF2, IF3) should be present for maximal activity

• The source of other components (tRNAs, elongation factors, release factors) should be carefully controlled and documented

Functional Readouts:

• Translation efficiency can be measured by monitoring the synthesis of full-length proteins

• The pattern of proteins produced may depend on the source of the 30S ribosomal subunit

Genetic Complementation:

• T. thermophilus IF2 has been shown capable of complementing E. coli infB mutants, providing an in vivo functional assay

• Growth rates and protein synthesis rates should be carefully monitored

Control Experiments:

• Parallel experiments with homologous systems (all components from the same organism)

• Systematic testing of hybrid systems with different combinations of components

• Inclusion of proper negative controls (factor-depleted systems)

Research has shown that "translation is indeed possible in such an in vitro chimeric system" combining components from T. thermophilus and E. coli, opening opportunities for detailed mechanistic studies of cross-species compatibility .