Recombinant Idiomarina loihiensis ATP-dependent RNA helicase rhlB (rhlB)

What is the function of RhlB in Idiomarina loihiensis?

RhlB in Idiomarina loihiensis, like other DEAD-box RNA helicases, is involved in RNA degradation mechanisms. It possesses RNA-dependent ATPase activity and catalyzes the unwinding of double-stranded RNA structures, facilitating RNA processing and degradation . As Idiomarina loihiensis is a halophilic γ-Proteobacterium isolated from the Lo'ihi submarine volcano in Hawaii , its RhlB likely plays a crucial role in RNA metabolism under the extreme conditions of its marine habitat.

What are the conserved motifs in RhlB essential for its catalytic activity?

RhlB contains several conserved motifs characteristic of DEAD-box helicases:

These motifs coordinate ATP binding, hydrolysis, and RNA interactions necessary for the helicase function . Mutations in these conserved regions typically result in loss of catalytic activity.

What expression systems are optimal for producing recombinant I. loihiensis RhlB?

While no specific expression system for I. loihiensis RhlB is detailed in the search results, several approaches can be recommended based on similar proteins:

• E. coli-based expression systems: Using pET vectors in BL21(DE3) strains with T7 RNA polymerase control.

• Cold-adapted expression: Since I. loihiensis is psychrophilic, expression at lower temperatures (15-20°C) may improve protein folding.

• Solubility enhancers: Fusion tags like MBP (maltose-binding protein) can improve solubility.

Expression conditions should be optimized considering the halophilic nature of I. loihiensis, potentially incorporating salt in the growth media to mimic its native environment .

What purification strategy yields the highest activity for recombinant RhlB?

A multi-step purification strategy is recommended:

• Initial capture: Affinity chromatography using His-tag or another suitable fusion tag

• Intermediate purification: Ion exchange chromatography (typically anion exchange)

• Polishing: Size exclusion chromatography

Critical considerations:

• Maintain elevated salt concentration (250-500 mM NaCl) in buffers to preserve structure and activity

• Include ATP or non-hydrolyzable ATP analogs during purification to stabilize the protein

• Add reducing agents (DTT or TCEP) to prevent oxidation of cysteine residues

• Use protease inhibitors to prevent degradation

Activity assays should be performed after each purification step to track retention of enzymatic function .

What methods are most reliable for measuring RhlB helicase activity?

Several complementary approaches can be used to measure RhlB helicase activity:

• Fluorescence-based unwinding assays: Using fluorescently labeled RNA duplexes with quencher pairs that separate upon unwinding.

• Real-time NMR spectroscopy: Monitoring structural changes in RNA substrates during unwinding, particularly useful for observing duplex opening in the absence of ATP as observed with E. coli RhlB .

• ATPase activity assays: Measuring ATP hydrolysis rates using:

  • Malachite green phosphate detection

  • 31P NMR real-time measurements

  • Coupled enzymatic assays with ATP regeneration

• RNA binding assays: Determining RNA substrate affinity through:

  • Fluorescence anisotropy

  • Electrophoretic mobility shift assays (EMSA)

  • 1H NMR titration experiments to monitor imino proton resonances

Each method provides different insights into the mechanistic aspects of RhlB function.

How does temperature affect RhlB activity from I. loihiensis?

As I. loihiensis is a marine psychrophilic bacterium, its RhlB likely exhibits cold-adaptation features:

• Temperature optima are expected to be lower than mesophilic homologs, potentially showing significant activity at 4-15°C.

• Activity characterization should include:

  • Temperature-dependent ATPase activity profiles (5-40°C range)

  • RNA unwinding rates at various temperatures

  • Thermal stability measurements (DSF or CD spectroscopy)

• Comparative analysis with E. coli RhlB would be valuable to identify cold-adaptation strategies in the enzyme structure and mechanism.

Studies with other psychrophilic bacteria like Pseudomonas aeruginosa and Caulobacter crescentus suggest DEAD-box helicases play important roles in cold adaptation mechanisms , and similar functions may exist in I. loihiensis.

Does I. loihiensis RhlB interact with other proteins to form a degradosome complex?

Based on studies of RhlB in other organisms, particularly E. coli, I. loihiensis RhlB likely participates in a degradosome complex:

• The primary interaction partner would be RNase E, as seen in E. coli where this interaction boosts RhlB's ATPase activity by an order of magnitude .

• Methods to investigate these interactions include:

  • Co-immunoprecipitation assays

  • Bacterial two-hybrid systems

  • Pull-down assays with recombinant proteins

  • Cross-linking coupled with mass spectrometry

• The specific characteristics of the I. loihiensis degradosome may differ from E. coli given its adaptation to marine environments and potentially different RNA processing requirements .

How can the interaction between RhlB and RNase E be characterized in I. loihiensis?

To characterize the RhlB-RNase E interaction in I. loihiensis:

• Interaction domain mapping: Using limited proteolysis, domain cross-linking and homology modeling approaches as performed for E. coli RhlB .

• Functional assays: Measuring ATPase and unwinding activities in the presence of:

  • Full-length RNase E

  • RNase E fragments containing the putative RhlB-binding region

  • RNase E with mutations in the binding interface

• Structural studies: NMR spectroscopy and X-ray crystallography of the complex to determine:

  • Binding interface residues

  • Conformational changes upon complex formation

  • Allosteric effects on catalytic domains

For E. coli, the interaction involves RhlB's carboxy-terminal RecA-like domain engaging a segment of RNase E that is approximately 64 residues long , which might be conserved in I. loihiensis.

What is the substrate specificity of I. loihiensis RhlB compared to other bacterial helicases?

The substrate specificity of I. loihiensis RhlB can be assessed through:

• Comparative binding and unwinding assays using:

  • RNA duplexes with different topologies (blunt-ended, 5'-overhang, 3'-overhang)

  • Single-stranded RNAs of varying lengths

  • Structured RNA elements (stem-loops, pseudoknots)

• RNA topology preference analysis: In E. coli, RhlB shows preference for 5'-extended duplexes over blunt-ended or 3'-extended substrates when interacting with RNase E .

• HITS-CLIP (High-throughput sequencing of RNA by cross-linking immunoprecipitation): To identify natural RNA targets of RhlB in vivo, as has been done with RhlB from Caulobacter crescentus where 220 transcripts were identified as binding partners .

The results would provide insights into how I. loihiensis RhlB is adapted to function in its specific cellular context.

How does RNA sequence and structure influence RhlB activity?

RNA sequence and structure significantly impact RhlB activity as demonstrated in studies with E. coli RhlB:

• RNA structural elements:

  • 5'-overhang duplexes show partial unwinding even in the absence of ATP when RhlB is bound to RNase E

  • Single-stranded regions facilitate initial binding

• Sequence preferences:

  • Potential preference for AU-rich regions (common in DEAD-box helicases)

  • GC-rich regions may require higher ATP consumption for unwinding

• Methodological approach:

  • Design RNA substrates with systematic variations in sequence composition

  • Use 13C-HSQC NMR spectroscopy to monitor conformational transitions in RNA upon protein binding

  • Correlate unwinding rates with RNA stability parameters

These studies would reveal how I. loihiensis RhlB has adapted to process RNA structures typical of its native environment.

How does RhlB contribute to cold adaptation in I. loihiensis?

Given that I. loihiensis is a marine bacterium that may experience cold environments, RhlB likely contributes to cold adaptation through:

• RNA structure modulation: Unwinding stabilized RNA structures that form at low temperatures.

• Cold-shock response: Potentially upregulated expression at lower temperatures, similar to findings in Caulobacter crescentus where the rhlB null mutant showed a freezing-sensitive phenotype .

• Ribosome assembly: May assist in RNA processing required for ribosome biogenesis at low temperatures.

Experimental approach:

• Compare growth phenotypes of wild-type and rhlB knockout strains at various temperatures

• Conduct transcriptomic analyses at optimal and low temperatures

• Measure global RNA decay rates at different temperatures in wild-type versus rhlB mutant strains

These analyses would reveal whether I. loihiensis RhlB, like its counterpart in C. crescentus, is essential for cryotolerance response .

What is the mechanism of ATP-independent RNA unwinding by RhlB when bound to RNase E?

Recent research with E. coli RhlB revealed the surprising finding that RhlB in complex with RNase E can induce conformational changes in RNA duplexes even in the absence of ATP . To investigate if I. loihiensis RhlB shares this property:

• Structural analysis:

  • Use 13C-HSQC NMR spectroscopy to detect partial duplex opening in the absence of ATP

  • Map the specific nucleotides undergoing conformational changes

• Mutational studies:

  • Generate point mutations in conserved motifs of RhlB to identify residues critical for ATP-independent activity

  • Create chimeric proteins between I. loihiensis and E. coli RhlB to map domains responsible for this activity

• Proposed model development:

  • Integrate structural and functional data to propose a mechanism for how RNase E binding alters RhlB's interaction with RNA substrates

  • Compare with the model developed for E. coli RhlB where the C-terminal domain interaction with RNase E leads to altered RNA binding

This would represent a significant advance in understanding the diverse mechanisms employed by DEAD-box helicases.

How does the genomic context of rhlB in I. loihiensis compare to other marine bacteria?

Analyzing the genomic context of rhlB in I. loihiensis provides insights into its evolution and specialized functions:

• Comparative genomic analysis:

  • Compare with other marine bacteria like Photobacterium profundum

  • Analyze synteny with psychrophilic γ-proteobacteria

  • Identify co-regulated genes that may be functionally related

• Regulatory elements:

  • Characterize promoter regions and transcription factor binding sites

  • Identify potential cold-responsive elements in the promoter region

• Evolutionary analysis:

  • Phylogenetic analysis of RhlB across marine bacteria

  • Identification of selective pressure signatures that might indicate functional adaptation

Similar analyses with other marine bacteria revealed specialized adaptations related to high salt conditions, cold temperatures, and pressure adaptations , which might also apply to I. loihiensis RhlB.

What are the best approaches for generating and characterizing rhlB mutants in I. loihiensis?

To generate and characterize rhlB mutants in I. loihiensis:

• Mutagenesis strategies:

  • Site-directed mutagenesis for targeting specific residues

  • CRISPR-Cas9 genome editing for chromosomal modifications

  • Construction of plasmid-based complementation systems

• Phenotypic characterization:

  • Growth curves at different temperatures (4°C, 15°C, 30°C)

  • Freezing sensitivity assays similar to those used for C. crescentus

  • RNA degradation rate measurements

• Molecular characterization:

  • Global transcriptome analysis (RNA-seq) comparing wild-type and mutant strains

  • Determination of RNA decay rates using rifampicin treatment followed by RNA-seq

  • Analysis of 5'-end transcripts to identify RNA degradation intermediates

The experimental design should include controls for complementation with wild-type rhlB to confirm phenotype specificity.

How can high-throughput methods be applied to study RhlB function in vivo?

High-throughput approaches for studying I. loihiensis RhlB function include:

• HITS-CLIP (High-throughput sequencing of RNA by cross-linking immunoprecipitation):

  • Express FLAG-tagged RhlB in I. loihiensis

  • Cross-link RNA-protein complexes with formaldehyde

  • Immunoprecipitate RhlB-RNA complexes

  • Sequence bound RNAs to identify targets

• RNA decay profiling:

  • Perform time-course RNA-seq after transcription inhibition

  • Compare decay rates between wild-type and rhlB mutant

  • Focus on structured RNAs that might require helicase activity

• Ribosome profiling:

  • Analyze impact of RhlB on translation efficiency

  • Identify mRNAs with altered ribosome occupancy in mutants

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and RNA structure probing

  • Develop computational models predicting RhlB targets based on RNA features

These approaches would provide comprehensive insights into the cellular functions of RhlB in I. loihiensis.