Recombinant Pseudomonas putida ATP-dependent RNA helicase rhlB (rhlB)

What is the ATP-dependent RNA helicase RhlB in Pseudomonas putida?

RhlB in Pseudomonas putida is an ATP-dependent RNA helicase that functions as a molecular motor involved in RNA metabolism. It belongs to the DEAD-box family of helicases that utilize ATP hydrolysis to unwind structured RNA molecules . The protein shares significant homology with RhlB in other bacterial species like Escherichia coli, where it has been more extensively characterized as a component of the RNA degradosome, a multi-enzyme complex involved in RNA processing and degradation . In bacterial systems, RhlB plays a crucial role in facilitating RNA degradation by unwinding structured regions that might otherwise impede the activity of ribonucleases.

How is the rhlB gene organized in the Pseudomonas genome?

In many bacteria, including Pseudomonas species, the genomic organization of rhlB appears to be conserved within specific contextual relationships with other genes. While not directly addressed for P. putida in the search results, comparative genomics studies indicate that in Pseudomonas aeruginosa, rhlB is associated with RNA degradosome components, similar to the organization observed in E. coli . It's important to note that the term "rhl" appears in multiple genetic contexts in Pseudomonas species - with the rhlABRI genes involved in rhamnolipid biosynthesis representing a distinct system from the RNA helicase rhlB , despite the similar nomenclature.

What protein domains are present in the rhlB helicase?

RhlB contains characteristic domains found in DEAD-box RNA helicases. Based on structural analyses, RhlB possesses a carboxy-terminal RecA-like domain that is crucial for protein-protein interactions, particularly with RNase E . In E. coli, this domain engages a segment of RNase E that is no greater than 64 residues in length . The protein likely also contains nucleotide-binding motifs for ATP interaction and hydrolysis, as well as RNA-binding regions that enable it to engage with target RNA substrates. The conserved domains allow RhlB to undergo conformational changes during the ATP binding and hydrolysis cycle that drive its RNA unwinding activity.

How does the interaction between rhlB and RNase E affect helicase activity?

The interaction between RhlB and RNase E significantly enhances the functional capabilities of the helicase. Studies in E. coli have demonstrated that this interaction boosts RhlB's ATPase activity by approximately an order of magnitude . The molecular basis for this activation involves two primary mechanisms:

• Direct stimulation: The binding of RNase E to RhlB's carboxy-terminal RecA-like domain directly stimulates both the unwinding and ATPase activities of the helicase .

• Spatial coordination: The interaction positions RhlB in proximity to RNA-binding sites on RNase E, allowing cooperative RNA targeting and unwinding .

Recent research has revealed that RNase E binding increases RhlB's affinity for specific RNA substrates, which contributes to higher ATP turnover rates . Most remarkably, in the presence of RNase E (residues 694-790), RhlB can induce conformational changes in RNA duplexes with 5'-overhangs even without ATP, leading to partial duplex opening . This represents a unique activation mode among DEAD-box helicases, as ATP binding is typically considered essential for RNA unwinding.

What structural changes occur in rhlB upon RNase E binding?

When RhlB interacts with RNase E, it undergoes structural alterations that enhance its catalytic properties. Although the complete structural transformation has not been fully elucidated, research indicates that binding of RNase E to RhlB's carboxy-terminal RecA-like domain triggers conformational changes that improve both RNA binding and ATP hydrolysis .

In P. aeruginosa, RhlB directly interacts with RNase E via the AR1 SLiM (RPRRRSRGQRRRSNRRERQR) region . Mutation of this AR1 sequence disrupts not only the RhlB-RNase E binding in vitro but also affects RhlB's subcellular localization and co-localization with RNase E in vivo . This suggests that the interaction induces significant changes in RhlB's spatial organization within the cell.

Interestingly, while in E. coli the AR1 SLiM primarily mediates RNA interaction, in P. aeruginosa it serves a dual role - contributing to both RNA binding and RhlB interaction . This functional difference highlights the evolutionary adaptations of this protein interaction network across bacterial species.

How does ATP binding and hydrolysis regulate rhlB activity?

In the presence of RNase E, RhlB demonstrates an enhanced ability to bind ATP and accelerated rates of ATP hydrolysis . This increased ATPase activity correlates directly with improved RNA unwinding capabilities. The mechanism appears to involve allosteric effects, where RNase E binding alters the conformational dynamics of RhlB's ATP-binding pocket, increasing its affinity for the nucleotide and/or accelerating steps in the hydrolysis reaction .

Most notably, recent research has revealed that in the presence of RNase E (694-790), RhlB can induce conformational changes in RNA duplexes with 5'-overhangs even in the absence of ATP, leading to partial duplex opening . This represents a significant departure from the conventional understanding of DEAD-box helicase mechanisms, where ATP binding is considered an essential prerequisite for RNA unwinding.

What assays can be used to measure rhlB-mediated RNA unwinding?

Several experimental approaches can be employed to assess the RNA unwinding activity of RhlB:

• Fluorescence-based assays: RNA substrates labeled with fluorophore-quencher pairs that exhibit changes in fluorescence upon unwinding can provide real-time measurements of helicase activity.

• Gel-based unwinding assays: Using native polyacrylamide gel electrophoresis to separate double-stranded and single-stranded RNA components after incubation with RhlB under various conditions.

• NMR spectroscopy: This technique has been successfully applied to study RhlB-induced conformational changes in RNA substrates with different topologies . NMR provides detailed structural information about the RNA during the unwinding process.

• RNA-centered approaches: Methodologies that focus on changes in RNA conformation rather than protein activity have proven valuable for understanding RhlB function. These approaches have revealed that RhlB exhibits different activities depending on the RNA substrate topology and the presence of RNase E .

When designing these assays, it's crucial to consider the impact of RNase E, as RhlB's activity is significantly enhanced when in complex with this partner protein. Including controls with and without RNase E (or relevant fragments) is essential for comprehensive analysis .

How can protein-protein interactions between rhlB and other components be analyzed?

Several techniques have been successfully employed to study RhlB's interactions with partner proteins:

• Bacterial two-hybrid (BTH) assay: This approach has been effective for verifying interactions between RhlB and RNase E. For example, co-expression of T25-RhlB with T18-RNase E resulted in a significant increase in β-galactosidase activity (5-fold increase) compared to negative controls, confirming their interaction .

• Pull-down assays: In vitro pull-down experiments using tagged proteins have demonstrated direct binding between RhlB and RNase E. For instance, RhlB was retained on beads containing Flag-RNase E 572-1057 but not on Flag-RNase E 1-529, and point mutations in the AR1 SLiM abolished this binding .

• Domain mapping through truncations: Creating a series of truncated versions of interaction partners has helped identify specific binding regions. This approach revealed that the AR1 SLiM in RNase E is crucial for RhlB binding .

• Fluorescence microscopy with tagged proteins: Visualizing the co-localization of fluorescently tagged RhlB with potential partners provides in vivo evidence of interactions. Studies showed that RhlB-msfGFP displays diffuse cytosolic localization in strains with truncated RNase E or mutations in the AR1 region, highlighting the importance of these elements for proper localization .

• Limited protease digestion and domain cross-linking: These methods have been useful for refining interaction sites between RhlB and RNase E, particularly in identifying that RhlB's carboxy-terminal RecA-like domain engages a specific segment of RNase E .

What are the optimal conditions for expressing recombinant P. putida rhlB in heterologous systems?

For successful expression of recombinant P. putida rhlB, several factors should be considered:

• Expression system selection: E. coli is commonly used for expressing recombinant proteins from Pseudomonas, though expression in P. putida itself may provide advantages for proper folding and modification of native proteins.

• Promoter selection: Strong inducible promoters like Tac have been successfully employed for expressing recombinant genes in P. putida. Studies on rhamnolipid-related genes demonstrated that replacing original promoters with the Tac promoter significantly increased expression levels, with 3-10 fold higher expression compared to native promoters .

• Codon optimization: Adapting the codon usage to the expression host can improve translation efficiency, particularly when expressing across different bacterial species.

• Vector design: Considering compatibility with the host strain and appropriate antibiotic resistance markers is essential. Successful recombinant expression has been achieved using vectors like p2-rhlAB-Tac in P. putida .

• Expression conditions: Optimizing parameters such as temperature, induction timing, and media composition is critical. The specific conditions would need to be determined empirically for rhlB, but successful expression of other recombinant proteins in P. putida provides useful starting points.

• Verification of expression: Quantitative RT-PCR has been effectively used to confirm the expression of recombinant genes in P. putida, allowing comparison of expression levels between different constructs and conditions .

How can the kinetics of rhlB-mediated RNA unwinding be quantified?

Quantifying the kinetics of rhlB-mediated RNA unwinding requires multiple analytical approaches to capture the complex nature of this ATP-dependent process:

• ATPase activity measurements: Since RNA unwinding by RhlB is coupled to ATP hydrolysis, measuring ATPase rates provides indirect insights into unwinding activity. This can be done using colorimetric assays for phosphate release or coupled enzyme assays that monitor ATP consumption.

• Time-course unwinding assays: By stopping reactions at different time points and analyzing the proportion of unwound RNA substrates, researchers can derive rate constants for the unwinding process.

• Single-molecule techniques: These approaches allow direct observation of individual unwinding events, providing detailed kinetic information that may be obscured in bulk experiments.

• Mathematical modeling: The kinetic data can be fitted to appropriate models to extract parameters such as:

  • kcat (catalytic rate constant)

  • Km for ATP

  • Binding affinities for RNA substrates

  • Rate-limiting steps in the reaction

When analyzing RhlB kinetics, it's critical to account for the significant impact of RNase E on its activity. Studies have shown that RNase E binding increases both the ATPase and unwinding activities of RhlB by an order of magnitude . This activation involves increased affinity for certain RNA substrates and enhanced ATP turnover rates .

The table below summarizes key kinetic parameters that would typically be measured for comprehensive characterization of rhlB activity:

How should contradictory results in rhlB functional studies be reconciled?

When confronting contradictory findings in rhlB research, several methodological approaches can help reconcile discrepancies:

• Experimental context evaluation: Differences in experimental conditions, protein preparations, or assay systems can significantly impact results. For instance, the presence or absence of single-strand binding proteins and ATP dramatically affects RecG (another bacterial helicase) activity on DNA substrates , and similar factors likely influence rhlB studies.

• Species-specific variations: Research indicates functional differences in rhlB between bacterial species. In P. aeruginosa, the AR1 SLiM of RNase E serves both RNA binding and RhlB interaction roles, while in E. coli it primarily mediates RNA interaction . Such species-specific adaptations can explain apparently contradictory findings across organisms.

• Substrate-dependent behavior: RhlB shows different activities depending on RNA substrate topology . Systematically testing a range of RNA structures with varying features (length, stability, overhangs) can help resolve contradictions related to substrate preferences.

• Protein partner effects: The dramatic enhancement of RhlB activity by RNase E binding means that the presence, absence, or concentration of this partner can create seemingly contradictory results. Studies should carefully control and report the status of relevant protein partners.

• Integration through structural biology: Techniques like NMR spectroscopy have revealed novel insights into RhlB mechanism, such as its ability to induce partial duplex opening even without ATP when RNase E is present . This finding helps reconcile contradictory observations about ATP dependence.

What are promising areas for advancing our understanding of rhlB function?

Several emerging research avenues hold potential for deepening our knowledge of rhlB:

• Structural characterization: Obtaining high-resolution structures of P. putida rhlB, both alone and in complex with RNase E and RNA substrates, would provide crucial insights into its activation mechanism. Particularly interesting would be capturing the conformational changes induced by RNase E binding that enable ATP-independent partial unwinding .

• Comparative studies across species: Systematic comparison of rhlB function across different Pseudomonas species and other bacteria could reveal evolutionary adaptations in RNA metabolism pathways. The observed differences in AR1 SLiM function between P. aeruginosa and E. coli highlight the value of such comparisons .

• In vivo RNA targeting: Developing methods to identify the specific RNA targets of rhlB in P. putida cells would connect biochemical activities to physiological functions. Techniques like CLIP-seq (crosslinking immunoprecipitation followed by sequencing) could reveal the RNA substrate landscape.

• Regulation of rhlB activity: Investigating how cellular factors beyond RNase E modulate rhlB function could uncover regulatory networks controlling RNA metabolism. Factors like Mg²⁺ concentration, which has been shown to have an opposite effect to ATP on RecG helicase activity , may play similar roles for rhlB.

• Integration with stress response: Exploring the role of rhlB in bacterial stress responses, particularly oxidative stress, represents an intriguing direction. The genomic organization of oxyR and recG helicases in an operon in many bacteria, including Pseudomonas species , suggests potential coordination between helicases and stress response systems that might extend to rhlB.