Recombinant Caulobacter sp. Translation initiation factor IF-2 (infB), partial

What is Translation Initiation Factor IF-2 (infB) and what is its role in bacterial protein synthesis?

Translation Initiation Factor 2 (IF2) is a key bacterial protein involved in the initiation phase of protein synthesis, which represents a crucial regulatory step in gene expression. IF2 performs several essential functions in translation initiation:

• Facilitates the binding of initiator tRNA (fMet-tRNA) and mRNA to the small (30S) ribosomal subunit to form the 30S initiation complex (30S IC)

• Mediates the association of the large (50S) ribosomal subunit with the 30S initiation complex

• Acts as a GTPase, with GTP hydrolysis being stimulated by the large ribosomal subunit, triggering the release of IF2 from the ribosome

• Positions ribosomal subunits in a distinct rotational orientation during the subunit-joining step of initiation

• Works cooperatively with other initiation factors (IF1 and IF3) to maintain the fidelity of start codon selection and initiator tRNA binding

These functions collectively ensure accurate initiation of protein synthesis at the correct start codon with the appropriate initiator tRNA. The initiation phase is particularly important as it establishes the reading frame for the subsequent elongation phase of translation.

How is the infB gene structured in bacteria, including Caulobacter species?

The infB gene, which encodes the IF2 protein, shows a characteristic pattern of conservation and variation across bacterial species:

• Within a single bacterial species, the infB gene generally shows limited intraspecies diversity, as demonstrated in studies of Streptococcus agalactiae

• When comparing across different bacterial species, the gene reveals distinctive patterns of conservation and variation in different regions

• The central and C-terminal parts of the gene are highly conserved across species, reflecting their critical functional roles

• The N-terminal part is highly variable in both length and amino acid sequence across different bacterial species

These structural features make the infB gene valuable for phylogenetic analyses. In S. agalactiae, researchers identified six different alleles of a partial sequence of infB among 58 genetically distinct strains . These alleles correlated with the separation of the same strains into major evolutionary lineages, demonstrating the utility of infB as a phylogenetic marker .

For Caulobacter specifically, resources such as CauloBrowser for Caulobacter crescentus provide genomic data that can be used to analyze the structure of the infB gene in this genus, though specific details about Caulobacter infB structure were not directly provided in the search results.

What are the key domains of the IF2 protein and their functions?

Based on comparative analysis of IF2 proteins from various bacterial species, the IF2 protein has a multi-domain architecture with distinct functional regions:

• N-terminal domain:

  • Highly variable in both length and amino acid sequence across bacterial species

  • This variability suggests species-specific functions or regulatory roles

  • May be involved in interactions with other cellular components specific to each bacterial species

• Central domain:

  • Highly conserved across bacterial species

  • Contains the GTP-binding site essential for IF2's GTPase activity

  • Critical for the core function of IF2 in translation initiation

  • Involved in ribosome interaction and subunit joining

• C-terminal domain:

  • Also highly conserved across bacterial species

  • Contains the region that interacts with the initiator tRNA

  • Essential for correctly positioning the initiator tRNA on the ribosome

  • Contributes to the specificity of IF2 for the initiator tRNA

The conservation of central and C-terminal domains across species underscores their essential roles in the fundamental process of translation initiation, while the variability in the N-terminal domain reflects potential adaptations to specific cellular environments or regulatory mechanisms in different bacterial species, including Caulobacter.

How does IF2 interact with ribosomes during translation initiation?

IF2 engages in complex interactions with the ribosome during translation initiation, as revealed by advanced studies using single-molecule FRET and cryo-EM:

• IF2 positions ribosomal subunits in a distinct rotational orientation during the subunit-joining step of translation initiation

• Specifically, IF2 stabilizes the ribosome in a "semirotated" conformation, which is intermediate between the fully rotated and nonrotated states

• This semirotated state shows a characteristic FRET value of 0.5, distinct from the nonrotated (0.6 FRET) and fully rotated (0.4 FRET) conformations

• IF2 also stabilizes the mobile domain of the large subunit called the L1 stalk in a unique half-closed position

• The interaction of IF2 with the ribosome is dependent on:

  • The presence of GTP or nonhydrolyzable GTP analogs (GDPCP, GDPNP, or GTPγS)

  • An aminoacylated initiator tRNA (fMet-tRNA^fMet)

• IF2 alone (without IF1 and IF3) is capable of inducing the semirotated conformation of the ribosome

• After GTP hydrolysis, IF2 dissociates from the ribosome, allowing the transition from the semirotated to the nonrotated conformation and progression to the elongation phase of protein synthesis

These ribosomal interactions are critical for proper formation of the translation initiation complex and subsequent protein synthesis, highlighting the central role of IF2 in bacterial translation.

What experimental methods are commonly used to study IF2 function and structure?

Several sophisticated experimental approaches have been employed to investigate IF2 function and structure:

• Single-molecule Fluorescence Resonance Energy Transfer (smFRET):

  • Crucial for studying the conformational dynamics of ribosomes during IF2-mediated initiation

  • By labeling the small and large ribosomal subunits with fluorophores (e.g., Cy3 and Cy5 on S6 and L9 proteins), researchers can monitor intersubunit rotation and conformational changes in real-time

  • smFRET revealed that IF2 stabilizes the ribosome in a semirotated conformation with a characteristic FRET value of 0.5

  • This approach allows detection of transient intermediates that might be missed in ensemble measurements

• Cryo-electron microscopy (Cryo-EM):

  • Provides visualization of structural details of IF2-bound ribosomes

  • Earlier cryo-EM reconstructions (>11 Å resolution) suggested that the small ribosomal subunit in 70S- IF2 ICs is rotated ~4-5° relative to the large ribosomal subunit

  • Higher resolution structures continue to reveal more detailed insights into IF2-ribosome interactions

• Stopped-flow kinetics:

  • Used to measure the kinetics of subunit association in the presence of initiation factors

  • Light scattering measurements detect the increase in particle size upon subunit joining

  • This method validated the activity of purified recombinant IFs and showed that IF2 accelerates subunit joining, whereas IF1 and IF3 significantly slow down subunit association in the absence of IF2

• Genetic and phylogenetic analyses:

  • Sequencing and alignment of the infB gene across multiple strains to assess diversity and evolutionary relationships

  • Revealed limited intraspecies diversity within S. agalactiae but significant interspecies variation, particularly in the N-terminal domain

  • Identified correlations between infB alleles and major evolutionary lineages

These complementary methods have provided multi-level insights into IF2 structure and function, from atomic-level details to larger-scale conformational changes and evolutionary patterns.

How does IF2 from Caulobacter sp. compare to IF2 from other bacterial species?

Based on the general patterns observed in bacterial IF2 proteins and the available information, we can infer several points about Caulobacter IF2 in comparison to other bacterial species:

• The IF2 protein generally shows a characteristic pattern of conservation and variability across bacterial species:

  • Highly conserved central and C-terminal domains that likely have identical core functions across species

  • A highly variable N-terminal domain in both length and amino acid sequence

• This pattern suggests that Caulobacter IF2 likely shares significant sequence similarity in its central and C-terminal domains with IF2 from other bacterial species, while potentially having a unique N-terminal domain that may reflect adaptations specific to Caulobacter's distinctive cell cycle and developmental program

• A comprehensive comparative analysis would typically involve:

  • Sequence alignment of Caulobacter IF2 with IF2 from well-studied model organisms like E. coli

  • Structural modeling to predict potential functional differences

  • Experimental validation through complementation studies or biochemical assays

• The phylogenetic utility of infB demonstrated in Streptococcus studies suggests that Caulobacter IF2 sequence analysis could provide insights into its evolutionary relationship with other bacterial species, particularly within the alpha-proteobacteria group

For researchers focused specifically on Caulobacter-specific features of IF2, systematic comparative sequence and functional analyses with IF2 proteins from diverse bacterial species would be particularly valuable.

What is known about the GTP hydrolysis mechanism of IF2 during translation initiation?

The GTP hydrolysis mechanism of IF2 serves as a critical molecular switch in translation initiation. Based on the research findings:

• IF2 is a translational GTPase that requires GTP for its function in facilitating subunit joining during initiation

• GTP hydrolysis is stimulated by the large ribosomal subunit (50S) upon its association with the 30S initiation complex

• This GTP hydrolysis triggers the release of IF2 from the ribosome, allowing transition to the elongation phase

• The transition from the semirotated to the nonrotated conformation of the ribosome is triggered by GTP hydrolysis/inorganic phosphate release or following IF2 disassociation from the ribosome

Experimental evidence has provided important insights into this mechanism:

• IF2 bound to nonhydrolyzable GTP analogs (GDPCP, GDPNP, or GTPγS) can still induce the semirotated conformation of the ribosome

• The predominant FRET value observed in subunit joining experiments with nonhydrolyzable GTP analogs was 0.5, indicating the semirotated conformation

• This suggests that the identity of the GTP analog does not influence IF2's ability to trap the ribosome in the semirotated conformation

• In contrast, IF2 in the absence of nucleotides or in the presence of GDP was unable to stabilize the semirotated conformation of the ribosome

• This observation is consistent with reports showing that IF2 dissociates from the ribosome upon GTP hydrolysis and promotes subunit joining much more efficiently with GTP or nonhydrolyzable GTP analogs than with GDP

These findings highlight how the GTPase activity of IF2 functions as a molecular timer controlling the progression of translation initiation.

How does IF2 contribute to translational fidelity in bacteria?

IF2 plays several critical roles in ensuring translational fidelity during the initiation phase of protein synthesis:

• Initiator tRNA selection:

  • IF2 specifically recognizes and binds to the initiator tRNA (fMet-tRNA^fMet)

  • This specificity helps prevent incorrect tRNAs from participating in initiation

  • The formation of the semirotated 70S initiation complex requires the presence of an aminoacylated initiator tRNA, implicating the IF2-mediated subunit joining step in preserving initiation fidelity

• Cooperative action with other initiation factors:

  • IF2 works together with IF1 and IF3 to cooperatively maintain the fidelity of start codon selection and initiator tRNA binding

  • This cooperative action provides multiple checkpoints to ensure accurate initiation

  • The factors work synergistically to reject incorrect tRNAs or mRNA start sites

• GTP-dependent conformational control:

  • GTP hydrolysis by IF2 controls the transition of the 70S initiation complex into the nonrotated conformation of the ribosome required for entry into the elongation phase

  • This ensures that elongation only proceeds after proper initiation complex formation

  • Serves as a timing mechanism to verify correct assembly before committing to translation

• Ribosomal subunit joining:

  • IF2 facilitates the correct association of the 50S ribosomal subunit with the 30S initiation complex

  • This process positions the ribosomal subunits in a specific rotational orientation (semirotated) that may serve as a checkpoint for proper initiation complex assembly

  • Helps ensure that only correctly formed initiation complexes progress to active translation

These mechanisms collectively ensure that translation begins at the correct start codon with the correct initiator tRNA, which is essential for accurate protein synthesis in bacteria including Caulobacter species.

What are the latest findings regarding IF2's role in ribosomal subunit joining and conformational changes?

Recent research has provided significant insights into IF2's role in ribosomal dynamics during translation initiation:

• IF2 stabilizes the ribosome in a "semirotated" conformation during the subunit-joining step of translation initiation, which is distinct from both the nonrotated and fully rotated states

• This semirotated state is characterized by a FRET value of approximately 0.5, intermediate between the nonrotated (0.6 FRET) and fully rotated (0.4 FRET) conformations

• The semirotated conformation represents a partial (~4-5°) rotation of the small ribosomal subunit relative to the large subunit

• This rotation is less than the degree of rotation observed in hybrid, fully rotated ribosomes

• IF2 alone (without IF1 and IF3) is capable of inducing this semirotated conformation of the ribosome

The stabilization of this unique conformational state requires specific conditions:

• The presence of GTP or nonhydrolyzable GTP analogs

• An aminoacylated initiator tRNA (fMet-tRNA^fMet)

• In contrast, IF2 in the absence of nucleotides or in the presence of GDP was unable to stabilize the semirotated conformation

Additional structural effects include:

• IF2 also stabilizes the mobile L1 stalk of the large ribosomal subunit in a unique half-closed position

• GTP hydrolysis triggered by the large ribosomal subunit leads to IF2 dissociation and subsequent transition of the ribosome from the semirotated to the nonrotated conformation

• This transition is necessary for the ribosome to enter the elongation phase of protein synthesis

These findings resolve earlier discrepancies in the literature regarding the conformational state of IF2-bound ribosomes and provide a clearer understanding of the structural dynamics during translation initiation.

How can single-molecule FRET be applied to study IF2-mediated ribosomal dynamics?

Single-molecule Fluorescence Resonance Energy Transfer (smFRET) has emerged as a powerful tool for studying IF2-mediated ribosomal dynamics. The following experimental design has been successfully implemented:

• Ribosomal subunit labeling:

  • Small (30S) ribosomal subunits are labeled with a donor fluorophore (e.g., Cy3) on protein S6

  • Large (50S) ribosomal subunits are labeled with an acceptor fluorophore (e.g., Cy5) on protein L9

  • This labeling strategy allows monitoring of intersubunit rotation as changes in FRET efficiency

• Sample preparation for initiation complex formation:

  • 30S initiation complexes (30S ICs) are assembled with:

    • Fluorescently labeled 30S subunits

    • Initiation factors (IF1, IF2, IF3)

    • GTP or nonhydrolyzable analogs

    • Initiator tRNA (fMet-tRNA^fMet)

    • mRNA with appropriate start codon

  • These complexes are tethered to a microscope slide using a biotinylated DNA oligonucleotide

• Real-time observation of subunit joining:

  • 30S ICs are initially imaged for a baseline period (e.g., 10 seconds)

  • Labeled 50S subunits are then injected into the sample chamber

  • The appearance of acceptor (Cy5) fluorescence indicates the joining of the 50S subunit

  • FRET efficiency is monitored in real-time to observe conformational changes

• Data analysis protocol:

  • FRET efficiency histograms are constructed from many individual ribosomes

  • Different FRET states correspond to different conformations:

    • 0.6 FRET = nonrotated conformation

    • 0.5 FRET = semirotated conformation (IF2-stabilized)

    • 0.4 FRET = fully rotated conformation

  • Kinetic information can be extracted from the time-resolved FRET traces

• Determining the effects of factors and conditions:

  • The influence of different components (e.g., IFs, GTP analogs) on ribosomal conformations can be assessed by systematically varying experimental conditions

  • For example, comparing results with GTP versus nonhydrolyzable GTP analogs reveals the role of GTP hydrolysis

This approach allows researchers to directly visualize the conformational dynamics of individual ribosomes during the initiation process, providing insights that would be difficult or impossible to obtain with ensemble techniques.

What are the challenges in expressing and purifying recombinant Caulobacter IF2 for structural studies?

While the search results don't directly address challenges specific to Caulobacter IF2 expression and purification, several potential challenges can be anticipated based on the properties of IF2 and general recombinant protein production considerations:

• Protein size and complexity challenges:

  • IF2 is a relatively large protein with multiple domains

  • The protein likely has a complex folding pathway, particularly given its multi-domain structure with both conserved and variable regions

  • The presence of a highly variable N-terminal domain may present unique folding challenges for Caulobacter IF2

• Expression system considerations:

  • Selecting an appropriate expression system is critical

  • E. coli is commonly used for bacterial protein expression, but codon usage differences between E. coli and Caulobacter might necessitate codon optimization

  • Alternative expression systems might be needed if E. coli cannot produce properly folded Caulobacter IF2

• Solubility and stability issues:

  • GTPases like IF2 often have solubility challenges due to their hydrophobic nucleotide-binding pockets

  • The presence of GTP or GTP analogs might be necessary to maintain stability during purification

  • Buffer optimization would be critical to prevent aggregation and maintain functionality

• Functional validation requirements:

  • Activity assays would be needed to confirm that the recombinant protein retains its GTPase activity

  • Verifying ribosome-binding capability would be essential, possibly using techniques like the light scattering assays mentioned in the research

• Structural heterogeneity challenges:

  • IF2 exists in different conformational states depending on nucleotide binding

  • This conformational flexibility might present challenges for structural studies requiring homogeneous samples

Methodological approaches to address these challenges might include fusion tags to enhance solubility, co-expression with chaperones, and careful optimization of expression conditions and purification protocols specific to Caulobacter IF2.

How might variations in the N-terminal domain of IF2 across bacterial species affect its function?

The N-terminal domain of IF2 shows significant variability across bacterial species in both length and amino acid sequence, while the central and C-terminal parts remain highly conserved . This pattern of conservation and variation suggests important functional implications:

Potential functional consequences of N-terminal domain variation:

• Species-specific interactions:

  • The variable N-terminal domain may mediate interactions with species-specific partners or regulatory factors

  • Such interactions could allow for fine-tuning of translation initiation in different bacterial cellular environments

  • These adaptations might reflect the diverse ecological niches occupied by different bacterial species

• Regulatory mechanisms:

  • The N-terminal domain might serve as a platform for species-specific regulatory mechanisms

  • Different bacterial species may have evolved distinct regulatory controls for translation initiation to respond to their particular environmental challenges

  • Post-translational modifications might target this variable region differently across species

• Ribosome binding adaptations:

  • While the core ribosome-binding function is likely preserved through the conserved domains, the variable N-terminus might adjust how IF2 interacts with ribosomes in different species

  • This could potentially influence the kinetics or stability of initiation complex formation

  • The semirotated conformation induced by IF2 might be subtly modulated by N-terminal variations

• Evolutionary significance:

  • The variability in the N-terminal domain makes it useful for phylogenetic analysis, as demonstrated in studies of Streptococcus agalactiae

  • This suggests that the N-terminal domain has been subject to different selective pressures compared to the conserved regions

  • The correlation between infB alleles and major evolutionary lineages in S. agalactiae indicates that N-terminal variations may reflect broader evolutionary adaptations

For Caulobacter specifically, understanding the unique features of its IF2 N-terminal domain could provide insights into how translation initiation might be adapted to its distinctive cell cycle and developmental program.

What are potential applications of recombinant Caulobacter IF2 in studying bacterial translation mechanisms?

Recombinant Caulobacter IF2 could serve as a valuable tool for various applications in bacterial translation research:

• Comparative structural and functional studies:

  • Comparing the structure and function of Caulobacter IF2 with IF2 from model organisms like E. coli could reveal conserved and divergent aspects of translation initiation

  • This could help identify fundamental principles of bacterial translation initiation versus species-specific adaptations

  • The single-molecule FRET approach described in the research could be applied to study whether Caulobacter IF2 induces similar ribosomal conformational changes as observed with E. coli IF2

• Exploration of unique Caulobacter cell cycle regulation:

  • Caulobacter is known for its asymmetric cell division and distinctive cell cycle

  • Investigating how IF2-mediated translation initiation might be regulated during different stages of the Caulobacter cell cycle could provide insights into the coordination of translation with cell division and differentiation

  • This could be particularly relevant since translation regulation is often coupled to cell cycle progression

• Phylogenetic and evolutionary studies:

  • As demonstrated with Streptococcus agalactiae, the infB gene can be useful for phylogenetic analyses

  • Characterizing Caulobacter IF2 could contribute to broader studies on the evolution of translation machinery in alpha-proteobacteria

  • The variable N-terminal domain of Caulobacter IF2 might provide insights into evolutionary adaptations specific to this bacterial group

• Systems biology integration:

  • The CauloBrowser mentioned in the search results is a systems biology resource for Caulobacter crescentus

  • Characterizing Caulobacter IF2 could contribute to the integration of translation regulation into systems-level models of Caulobacter biology

  • This could advance understanding of how translation is coordinated with other cellular processes in this model organism

• Mechanism of action studies:

  • Detailed investigation of how Caulobacter IF2 interacts with GTP and the ribosome

  • Examination of whether Caulobacter IF2 stabilizes the semirotated conformation of the ribosome as observed with E. coli IF2

  • Determination of kinetic parameters of GTP hydrolysis and ribosome binding specific to Caulobacter IF2

These applications would benefit from combining recombinant protein studies with advanced techniques such as cryo-EM, single-molecule FRET, and ribosome profiling to comprehensively characterize Caulobacter IF2 function within this important model organism's unique biological context.