Recombinant Staphylococcus aureus Ribonuclease J 1 (SAS1024), partial

What is Staphylococcus aureus Ribonuclease J1 and what are its basic properties?

Staphylococcus aureus Ribonuclease J1 (SAS1024) is a critical enzyme involved in RNA metabolism with dual nucleolytic activities. The protein functions as a manganese-dependent homodimeric enzyme exhibiting both 5′→3′ exoribonuclease and endoribonuclease activities. Encoded by the rnjA gene in S. aureus, RNase J1 plays essential roles in RNA turnover and maturation. The recombinant partial protein (product code CSB-MP739937SKW) is typically expressed in mammalian cells with >85% purity as determined by SDS-PAGE .

How does Staphylococcus aureus RNase J1 differ from RNase J in other bacterial species?

While RNase J is evolutionarily conserved across many bacterial species, S. aureus RNase J1 (SauJ1) exhibits distinct characteristics compared to orthologs in other organisms. Unlike Chlamydomonas reinhardtii RNase J, which demonstrates exclusively endonucleolytic activity in vitro, SauJ1 possesses robust dual activities. Bacillus subtilis RNase J1 functions primarily as an exonuclease in vitro, while Arabidopsis RNase J displays strong endonucleolytic activity with relatively minor exonucleolytic functions. Plant RNase J uniquely contains a GT-1 domain absent in bacterial counterparts, which may confer additional regulatory capabilities .

What are the optimal storage and handling conditions for recombinant S. aureus RNase J1?

Recombinant S. aureus RNase J1 stability is influenced by storage conditions, buffer composition, and temperature. For optimal preservation, store lyophilized protein at -20°C/-80°C, where it maintains stability for up to 12 months. Liquid formulations have a shorter shelf life of approximately 6 months at similar temperatures. When reconstituting the protein, use deionized sterile water to achieve concentrations between 0.1-1.0 mg/mL. Addition of glycerol (recommended final concentration 50%) enhances stability for long-term storage. Avoid repeated freeze-thaw cycles, and working aliquots can be stored at 4°C for up to one week .

What are the dual enzymatic activities of S. aureus RNase J1 and how can they be measured?

S. aureus RNase J1 exhibits both 5'→3' exoribonucleolytic and endoribonucleolytic activities. To measure these distinct functions:

Exoribonuclease activity assay:

• Use 5'-monophosphorylated RNA substrates labeled at either terminus

• Incubate with purified RNase J1 in buffer containing manganese (essential cofactor)

• Monitor degradation patterns via gel electrophoresis, distinguishing progressive 5'→3' degradation

• Quantify activity by measuring disappearance of full-length substrate or appearance of mononucleotide products

Endoribonuclease activity assay:

• Employ circularized RNA substrates (lacking free 5' ends) or RNA with protected 5' ends

• Observe internal cleavage patterns through gel electrophoresis

• Map specific cleavage sites using primer extension or RNA sequencing

These activities are manganese-dependent, with SauJ1 showing robust activity in both capacities unlike orthologs that may demonstrate preference for one activity .

How does the manganese dependence of S. aureus RNase J1 affect experimental design?

The manganese dependence of S. aureus RNase J1 is a critical factor in experimental design. When establishing in vitro assay conditions:

• Buffer composition should include optimal Mn²⁺ concentrations (typically 1-5 mM)

• Enzyme activity can be modulated by varying manganese levels

• EDTA and other metal chelators must be avoided as they will inhibit activity

• Control experiments should include metal-free conditions to confirm specificity

• Other divalent cations (Mg²⁺, Ca²⁺) may provide partial activity but with altered kinetics

This manganese dependence distinguishes RNase J1 from some other ribonucleases and provides a mechanism to specifically control its activity in reconstituted systems. Researchers should monitor manganese concentrations carefully as both insufficient and excessive levels can impact experimental outcomes .

How does S. aureus RNase J1 process 5' triphosphorylated RNA compared to monophosphorylated RNA?

Unlike many exoribonucleases that require monophosphorylated 5' ends, S. aureus RNase J1 can efficiently degrade 5' triphosphorylated RNA substrates. Experimental evidence shows:

• SauJ1 processes triphosphorylated RNAs with only modestly reduced efficiency compared to monophosphorylated substrates

• This distinguishes it from enzymes like RNase E that strongly prefer monophosphorylated substrates

• The ability to degrade triphosphorylated RNA directly expands its potential targets to include primary transcripts

This capability may explain why RNase J1 plays such a significant role in RNA turnover in S. aureus, as more than 70% of RNA fragments show increased abundance in RNase J1 deletion strains. For accurate activity assessment, researchers should test both tri- and mono-phosphorylated substrates when characterizing the enzyme's activity profile .

What are the major biological functions of RNase J1 in S. aureus RNA metabolism?

RNase J1 serves multiple critical functions in S. aureus RNA metabolism:

• Bulk RNA degradation: Acts as a primary enzyme in mRNA turnover pathways

• RNA maturation: Essential for 5' end processing of specific transcripts, including:

  • 16S ribosomal RNA maturation

  • RNase P ribozyme processing

• Quality control: Eliminates aberrant RNA species and antisense transcripts

• Gene expression regulation: Influences expression levels of approximately one-third of genes

Deletion studies demonstrate that while not absolutely essential, RNase J1 is required for growth under most conditions, with mutants restricted to narrow temperature and media ranges. RNA-seq data reveals that over 70% of RNA fragments show increased abundance in RNase J1 deletion strains, highlighting its central role in RNA turnover .

How does RNase J1 contribute to resolving stalled RNA polymerase complexes?

RNase J1 employs a "torpedo" mechanism to resolve stalled RNA polymerase (RNAP) complexes:

• When RNAP stalls during transcription, RNase J1 accesses the exposed 5' end of the nascent transcript

• It degrades the RNA in a 5'→3' direction, progressively advancing toward the stalled polymerase

• Upon reaching the RNAP, the enzyme causes complex disassembly and RNAP release from DNA

• This mechanism prevents potentially deleterious transcription-replication collisions

Experimental evidence from B. subtilis shows that RNase J1 is more efficient at this function than heterologous 5'→3' exonucleases like yeast Xrn1, suggesting species-specific optimization. ChIP-seq data demonstrates increased RNAP occupancy in RNase J1 deletion strains without corresponding increases in transcript levels, indicating accumulation of stalled, non-productive complexes .

How can recombinant S. aureus RNase J1 be used as a research tool in RNA biology?

Recombinant S. aureus RNase J1 offers several valuable applications as a research tool:

• RNA structure mapping:

  • Probing accessible 5' ends in complex RNA structures

  • Distinguishing between protected and exposed RNA regions

• RNA processing studies:

  • Reconstitution of in vitro RNA maturation pathways

  • Identification of sequence or structural determinants in RNA processing

• Transcription termination analysis:

  • Studying the "torpedo" mechanism of transcription termination

  • Investigating factors that enhance or inhibit RNAP dissociation

• Antisense RNA regulation:

  • Examining the role of RNase J1 in eliminating antisense transcripts

  • Understanding mechanisms of transcriptional interference

When using the recombinant protein as a tool, researchers should account for its dual nucleolytic activities and manganese dependence to properly interpret experimental results .

What is the optimal protocol for purifying active recombinant S. aureus RNase J1?

To purify active recombinant S. aureus RNase J1:

• Expression system:

  • Mammalian cell expression provides optimal folding and activity

  • Include a purification tag (typically His6 or Step-Flag) at the C-terminus to preserve activity

• Purification steps:

  • Lyse cells in buffer containing manganese and protease inhibitors

  • Perform affinity chromatography using the appropriate resin

  • Add intermediate ion exchange chromatography step for higher purity

  • Finish with size exclusion chromatography to obtain homogeneous dimeric enzyme

• Quality control:

  • Verify purity by SDS-PAGE (target >85%)

  • Confirm dimeric state by native PAGE or gel filtration

  • Validate activity using standard exo- and endoribonuclease assays

  • Store in buffer containing glycerol at -80°C

This methodology was successfully employed to produce the enzyme described in the literature with robust dual nucleolytic activities .

How can researchers establish in vitro assays to specifically measure RNase J1 exo- versus endo-nucleolytic activities?

To differentially assess RNase J1's exo- and endo-nucleolytic activities:

Exonuclease-specific assay:

• Prepare linear RNA substrates with 5'-monophosphate ends

• Label at 3' end to visualize degradation products

• Use non-structured RNA to prevent internal cleavage

• Analyze time-course by denaturing PAGE to observe progressive 5'→3' degradation pattern

• Quantify disappearance of full-length substrate

Endonuclease-specific assay:

• Block 5' end with strong secondary structure or chemical modification

• Alternatively, use circular RNA lacking free 5' ends

• Uniformly label substrate to visualize all cleavage products

• Analyze by denaturing PAGE to identify discrete cleavage products

• Map cleavage sites using primer extension or sequencing

Control experiments:

• Include catalytically inactive mutant (H74A-H76A) as negative control

• Compare activities under varying Mn²⁺ concentrations

• Test temperature dependence (typically 30-37°C optimal)

These approaches allow separate quantification of each activity using the same enzyme preparation, enabling structure-function studies and inhibitor screening .

What approaches can be used to study the in vivo targets and functions of RNase J1 in S. aureus?

To investigate in vivo targets and functions of RNase J1 in S. aureus:

• Genetic approaches:

  • Generate conditional depletion strains (as complete deletion may affect viability)

  • Create point mutations affecting specific activities (separating exo- from endo-activity)

  • Employ CRISPR interference for targeted repression

• Transcriptome analysis:

  • RNA-seq comparing wild-type and RNase J1-depleted strains

  • EMOTE (exact mapping of transcriptome ends) to identify processing sites

  • Differential RNA-seq to distinguish primary from processed transcripts

• Protein-RNA interaction methods:

  • CLIP-seq (crosslinking immunoprecipitation) to identify direct RNA targets

  • Gradient fractionation to determine association with ribosomes or other complexes

  • In vivo RNA structure probing to identify RNase J1-dependent structural changes

• Functional validation:

  • Targeted reporter assays for specific transcript processing events

  • In vitro reconstitution with purified components

  • Pulse-chase experiments to measure altered RNA decay rates

These complementary approaches have revealed that RNase J1 processes specific transcripts like 16S rRNA and RNase P while also participating in bulk RNA turnover .

What structural features distinguish S. aureus RNase J1 from other bacterial ribonucleases?

S. aureus RNase J1 exhibits several distinctive structural features:

• Domain organization:

  • β-lactamase-like core domain containing the catalytic site

  • β-CASP domain involved in nucleic acid binding

  • C-terminal domain mediating dimerization

• Active site architecture:

  • Contains conserved histidine residues (H74, H76) essential for catalysis

  • Metal-binding pocket accommodating manganese ions

  • Unique substrate channel allowing 5'→3' progression

• Dimeric structure:

  • Forms obligate homodimers unlike many other ribonucleases

  • Dimerization is required for full enzymatic activity

  • Interface creates extended RNA binding surface

Unlike many bacterial RNases that are strictly endo- or exo-nucleases, RNase J1's structure enables dual activities within a single enzyme. The 5'→3' exonuclease activity is particularly notable as this directionality is relatively uncommon in bacterial systems, with most bacterial exoribonucleases operating in the 3'→5' direction .

How do mutations in the catalytic site affect the dual activities of RNase J1?

Mutations in the catalytic site of RNase J1 have differential effects on its dual enzymatic activities:

• Key catalytic residues:

  • The double alanine mutation (H74A-H76A) abolishes both activities

  • These conserved histidines coordinate the essential manganese ions

• Substrate binding pocket mutations:

  • Alterations to residues lining the 5' end binding pocket typically affect exonuclease activity more severely than endonuclease activity

  • Mutations in the RNA-binding path can modify the processivity of the exonuclease function

• Metal coordination sphere:

  • Changes to residues in the second coordination sphere of manganese can differentially impact the two activities

  • Such mutations can potentially create variants with enhanced specificity for one activity

This structure-function relationship provides opportunities to engineer variants with specialized activities for particular experimental applications. The H74A-H76A mutant serves as an invaluable negative control in biochemical assays assessing RNase J1 activity .

What is the molecular mechanism of the "torpedo effect" in transcription complex dissociation?

The molecular mechanism of RNase J1's "torpedo effect" in resolving stalled transcription complexes involves:

• Initial targeting:

  • RNase J1 recognizes and binds the 5' end of nascent RNA in stalled transcription complexes

  • The enzyme is physically linked to RNA polymerase (RNAP) through the RNA strand

• Degradation phase:

  • Progressive 5'→3' exonucleolytic degradation of the RNA occurs

  • RNase J1 advances toward the stalled RNAP along the RNA strand

• Collision and displacement:

  • Upon reaching proximity to RNAP, RNase J1 causes complex destabilization

  • The transcription bubble collapses as the RNA-DNA hybrid is disrupted

  • RNAP dissociates from the DNA template

• Mechanism specificity:

  • RNase J1 is more efficient at displacing B. subtilis RNAP than heterologous enzymes like yeast Xrn1

  • The effect extends to E. coli RNAP, suggesting conservation of the basic mechanism across bacteria

Experimental evidence shows that RNase J1 treatment results in release of RNAP from DNA templates in reconstituted systems, confirming the direct role of the enzyme in complex disassembly rather than just RNA degradation .

How does S. aureus RNase J1 compare functionally to plant RNase J with its GT-1 domain?

The functional comparison between S. aureus RNase J1 and plant RNase J reveals significant evolutionary distinctions:

The plant-specific GT-1 domain found in Arabidopsis RNase J potentially confers additional regulatory capabilities through sequence-specific binding or protein-protein interactions. This domain's structural conservation suggests it maintains functional significance despite its absence in bacterial enzymes like S. aureus RNase J1 .

What are the key differences in RNase J1 function between S. aureus and B. subtilis?

Although RNase J1 serves similar broad functions in S. aureus and B. subtilis, several key differences exist:

• Essentiality:

  • In B. subtilis, RNase J1 deletion strongly affects expression of one-third of genes

  • In S. aureus, RNase J1 is non-essential but its deletion restricts growth conditions

• RNA processing targets:

  • Both process 16S rRNA, but may have species-specific additional targets

  • S. aureus RNase J1 processes the RNase P ribozyme

• Genetic context:

  • In B. subtilis, RNase J1 is organized in a two-gene operon with rpoY (encoding RNAP subunit ε)

  • This genetic linkage suggests potential functional coordination with transcription

• Torpedo mechanism:

  • B. subtilis RNase J1 has been experimentally demonstrated to resolve stalled RNAP complexes

  • While likely conserved in S. aureus, this function has been more extensively characterized in B. subtilis

These differences highlight the evolutionary adaptation of RNase J1 to the specific RNA metabolism requirements of each bacterial species while maintaining core functions in RNA processing and decay .

How can researchers determine whether RNase J1 functions primarily as an exo- or endo-nuclease in vivo?

Determining the predominant in vivo activity of RNase J1 requires multiple complementary approaches:

• Transcriptome-wide RNA end mapping:

  • EMOTE (exact mapping of transcriptome ends) to identify 5' ends

  • Comparison between wild-type and RNase J1-depleted strains

  • Classification of ends as exonucleolytic (progressive shortening) or endonucleolytic (discrete sites)

• Variant complementation studies:

  • Generate separation-of-function mutations affecting only one activity

  • Express these variants in RNase J1-depleted backgrounds

  • Determine which activity rescues specific phenotypes

• In vivo RNA structure analysis:

  • SHAPE-seq or similar methods to identify RNase J1-dependent structural changes

  • Distinguish between internal cleavages and end trimming

• Substrate trapping approaches:

  • Catalytically inactive mutants to trap substrates

  • Analysis of bound RNAs to determine binding mode

What are the potential consequences of RNase J1 inhibition for bacterial physiology and pathogenesis?

Inhibition of RNase J1 in S. aureus would likely have multifaceted effects:

• Transcriptome disruption:

  • Global alterations in mRNA abundance and stability

  • Accumulation of incompletely processed transcripts

  • Changes in regulatory RNA levels and function

• Ribosome biogenesis defects:

  • Impaired 16S rRNA maturation affecting ribosome assembly

  • Potential reduction in translational capacity

  • Altered stress responses mediated by specialized ribosomes

• Genomic stability issues:

  • Increased frequency of stalled transcription complexes

  • Higher risk of transcription-replication collisions

  • Potential increase in mutation rates due to replication stress

• Virulence modulation:

  • Altered expression of virulence factors dependent on precise mRNA regulation

  • Changed stress adaptation capacity in host environments

  • Potential attenuation of pathogenesis through multiple pathways

How might researchers design specific inhibitors of S. aureus RNase J1 for potential therapeutic applications?

Designing specific inhibitors of S. aureus RNase J1 requires a multifaceted approach:

• Structure-based design:

  • Target the unique manganese-binding pocket

  • Focus on regions that differ from human ribonucleases

  • Develop compounds that block the RNA binding channel

  • Consider allosteric inhibitors disrupting dimerization

• High-throughput screening strategies:

  • Develop fluorescence-based activity assays suitable for large-scale screening

  • Use differential screening to identify compounds specific to RNase J1 versus related enzymes

  • Counter-screen against human ribonucleases to ensure selectivity

• Validation methodologies:

  • Test effects on purified enzyme (IC50 determination)

  • Evaluate cellular penetration and target engagement

  • Assess phenotypic effects matching RNase J1 genetic depletion

  • Examine transcriptome-wide effects using RNA-seq

• Optimization considerations:

  • Balancing potency, selectivity, and pharmacokinetic properties

  • Addressing potential resistance mechanisms

  • Combination strategies with traditional antibiotics

These approaches could yield novel antibacterial compounds with mechanisms distinct from current antibiotics, potentially addressing growing resistance concerns .

What experimental approaches could elucidate the interplay between RNase J1 and RNA polymerase during transcription-coupled RNA processing?

To investigate the dynamic relationship between RNase J1 and RNA polymerase:

• In vivo proximity mapping:

  • BioID or APEX2 proximity labeling fused to RNase J1

  • ChIP-seq with anti-RNase J1 antibodies to map genomic associations

  • NET-seq to identify nascent transcripts associated with both proteins

• Real-time single-molecule approaches:

  • Fluorescently labeled components in reconstituted systems

  • FRET-based detection of RNase J1-RNAP interactions

  • Direct visualization of "torpedo" events in real-time

• Structural biology:

  • Cryo-EM of RNase J1 bound to stalled transcription complexes

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • Cross-linking mass spectrometry to identify proximity relationships

• Genetic interaction mapping:

  • Synthetic genetic arrays testing interactions between RNase J1 and RNAP mutations

  • Suppressor screens to identify compensatory mutations

  • Targeted mutagenesis of potential interaction interfaces

These approaches would provide mechanistic insight into how RNase J1 recognizes and resolves stalled transcription complexes, potentially revealing novel principles in bacterial gene expression coordination .

What are the optimal reconstitution and storage conditions for maintaining RNase J1 activity in laboratory settings?

For maintaining optimal RNase J1 activity in laboratory settings:

• Reconstitution protocol:

  • Briefly centrifuge lyophilized protein vial before opening

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 50% for stability

  • Avoid buffers containing metal chelators (EDTA, EGTA)

  • Allow complete dissolution before activity testing

• Storage recommendations:

  • Lyophilized form: stable for 12 months at -20°C/-80°C

  • Liquid form: stable for 6 months at -20°C/-80°C

  • Working aliquots: store at 4°C for up to one week

  • Minimize freeze-thaw cycles (prepare single-use aliquots)

• Activity preservation:

  • Include Mn²⁺ in storage and reaction buffers (1-5 mM)

  • Monitor for activity decline over time with standard assays

  • Consider adding protease inhibitors for extended storage

  • Avoid oxidizing conditions that may affect metal coordination

Following these guidelines ensures reliable enzyme performance across experiments and maximizes the shelf life of this valuable research reagent .

How can researchers design RNA substrates to specifically study either the exo- or endo-nucleolytic activities of RNase J1?

To design RNA substrates that selectively probe specific RNase J1 activities:

For exonuclease activity studies:

• Design linear RNA with defined 5' ends (monophosphorylated preferred)

• Include a fluorescent label or radiolabel at the 3' end to track degradation

• Avoid strong secondary structures near the 5' end

• Use lengths of 50-200 nucleotides for optimal detection of progressive degradation

• Include control substrates with blocked 5' ends (triphosphate or cap structures)

For endonuclease activity studies:

• Create circular RNA substrates lacking free 5' ends

• Design RNAs with 5' ends protected by strong hairpins or chemical modifications

• Include internal structure motifs that might serve as recognition sites

• Use uniformly labeled substrates to detect all cleavage products

• Consider substrates with strategic internal fluorophore/quencher pairs

Comparative analysis substrates:

• Design structurally identical substrates differing only in 5' end chemistry

• Create chimeric substrates with regions from known natural targets

• Include binding competitors to assess specificity determinants

These substrate design principles enable precise characterization of each activity and facilitate structure-function studies of the enzyme's dual catalytic capabilities .

What experimental controls are essential when studying the molecular functions of recombinant RNase J1?

Essential experimental controls for studying recombinant RNase J1 include:

• Negative controls:

  • Catalytically inactive mutant (H74A-H76A double alanine mutant)

  • Heat-inactivated enzyme preparations

  • Reaction buffer lacking manganese

  • RNase-free conditions verified with RNase-sensitive substrates

• Positive controls:

  • Well-characterized RNA substrates with established degradation patterns

  • Commercial RNases with defined activities (RNase A, T1)

  • Previously validated enzyme preparation with known activity levels

• Specificity controls:

  • Substrates resistant to 5'→3' degradation (5'-blocked RNAs)

  • Substrates resistant to endonucleolytic cleavage (highly structured RNAs)

  • Competition with non-specific RNA to assess target preference

  • Varying manganese concentrations to confirm metal dependence

• Technical controls:

  • Time-course analysis to establish reaction kinetics

  • Enzyme titration to ensure linearity of response

  • Temperature controls to account for activity variation

  • Fresh vs. stored enzyme comparison to detect activity loss

These comprehensive controls ensure experimental rigor and reproducibility when investigating the complex dual activities of RNase J1 and help distinguish between direct enzymatic effects and potential contaminants or artifacts .