Recombinant Rhodopirellula baltica Glycine--tRNA ligase (glyQS), partial
Description
Structure and Function
Glycyl-tRNA synthetase (GlyRS) facilitates the crucial attachment of glycine to tRNA(Gly), a fundamental step in protein synthesis . In bacteria, GlyRS is typically composed of two subunits, α and β . The α-subunit contains the aminoacylation site, which is responsible for catalyzing the attachment of glycine to tRNA . The β-subunit contains important tRNA recognition elements .
Rhodopirellula baltica is a marine bacterium known for its unique cell structure and metabolic capabilities . The GlyRS from R. baltica shares the same function as GlyRS from other organisms, but it may have some unique structural features that are adapted to its specific cellular environment .
Classes of GlyRS
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Class I GlyRS Recognizes glycine using a distinct set of residues compared to Class II GlyRS .
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Class II GlyRS Employs different chemical strategies for glycine recognition and includes a new subclass IId, which contains AlaRS and bacterial α2β2 GlyRS .
Key Functions of GlyRS
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Aminoacylation: Attaching glycine to tRNA(Gly) is the primary function .
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Transcription Antitermination: Involved in tRNA-directed transcription antitermination of genes .
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Regulation: Influences RNA metabolism through glycine-rich RNA-binding proteins (GR-RBPs) .
Rhodopirellula baltica GlyQS
The glyQS gene encodes GlyRS in Bacillus subtilis . The glyQS leader region is involved in tRNA-directed antitermination, a process where the transcription of the glyQS gene is regulated by the availability of charged tRNA .
Regulation of glyQS
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tRNA-directed antitermination Transcription of glyQS is regulated by the interaction of tRNAGly with the glyQS leader region .
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NusA protein The addition of B. subtilis NusA to the glyQS antitermination reaction can result in increased termination in the absence of tRNA .
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Specifier sequence The interaction between the glyQS leader and tRNAGly is highly specific, with specific sequences in the leader and anticodon of tRNA being crucial for the interaction .
Research Findings
Structural analysis reveals that bacterial GlyRS α-subunit contains all the determinants for the first step of the reaction and can perform this catalysis . Key interactions for glycine recognition involve several conserved residues such as Trp-115, Gln-76, Gln-78, Thr-33, and Glu-156 .
Table 1: Conserved Residues in Bacterial GlyRS
These findings highlight the structural and functional diversity of GlyRS across different organisms and provide insights into the evolution and regulation of protein synthesis .
Glycine-Rich RNA-Binding Proteins (GR-RBPs)
Glycine-rich RNA-binding proteins (GR-RBPs) are a class of proteins that play critical roles in RNA metabolism . These proteins are characterized by a glycine-rich domain and one or more RNA-binding domains, such as an RNA recognition motif (RRM) or a cold-shock domain (CSD) . GR-RBPs are involved in various aspects of RNA processing, including alternative splicing, mRNA export, and RNA editing .
Functions of GR-RBPs
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RNA Processing GR-RBPs regulate RNA alternative splicing, polyadenylation, and miRNA biogenesis .
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Stress Response They mediate plant stress responses by influencing RNA stability and translation .
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Phase Separation The glycine-rich domain can drive phase separation, enhancing stress tolerance in plants .
Product Specs
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Target Background
Catalyzes the attachment of glycine to tRNA(Gly).
KEGG: rba:RB10547
STRING: 243090.RB10547
Q&A
What is the fundamental reaction catalyzed by R. baltica Glycine--tRNA ligase?
Rhodopirellula baltica Glycine--tRNA ligase (glyQS) catalyzes the same fundamental reaction as other glycyl-tRNA synthetases: the attachment of glycine to its cognate tRNA. The reaction follows the general equation:
ATP + glycine + tRNAGly → AMP + diphosphate + glycyl-tRNAGly
This two-step reaction first forms a glycyl-adenylate intermediate by activating glycine with ATP, followed by the transfer of the glycyl moiety to the 3' end of tRNAGly. This reaction is essential for protein synthesis as it provides the charged tRNA needed for translating glycine codons during mRNA translation . The enzyme belongs to the ligase family that forms carbon-oxygen bonds in aminoacyl-tRNA and related compounds, with the systematic name being glycine:tRNAGly ligase (AMP-forming) .
How does R. baltica glyQS recognition of tRNA differ from other bacterial species?
R. baltica glyQS likely follows a two-step binding model similar to that observed in T-box riboswitches that recognize tRNAGly. In this model, the anticodon of tRNAGly is recognized first, creating a partially bound state, followed by binding of the tRNA 3' NCCA end to form a fully bound state . This process involves significant conformational changes in both the enzyme and tRNA molecule during the glycylation process, similar to what has been observed with human glycyl-tRNA synthetase .
The distinctive cell biology of R. baltica, with its morphological transitions throughout its life cycle, suggests potential growth phase-dependent regulation of glyQS activity. Gene expression studies of R. baltica have shown significant transcriptional changes throughout different growth phases, which may affect aminoacyl-tRNA synthetase expression and activity .
What structural domains are critical for R. baltica glyQS function?
Based on research on other glycyl-tRNA synthetases, R. baltica glyQS likely contains several critical structural domains:
| Domain | Predicted Function | Conservation Level |
|---|---|---|
| Active Site | Glycine and ATP binding | Highly conserved |
| Anticodon Binding Domain | tRNA anticodon recognition | Moderately conserved |
| Insertion Domains | Facilitating tRNA binding | Variable across species |
| Dimerization Interface | Stabilizing quaternary structure | Conserved in class II aaRS |
Studies of human glycyl-tRNA synthetase have shown that insertions 1 and 3 work cooperatively with the active site to facilitate efficient substrate binding . Mutational studies in bacterial glycyl-tRNA synthetases have demonstrated that even single amino acid changes, such as a Pro-61→Leu substitution in the alpha chain, can significantly alter substrate binding affinity, increasing the Km values for glycine (25-fold) and ATP (45-fold) while having minimal effects on tRNA binding .
How do conformational changes impact R. baltica glyQS catalytic efficiency?
Structural studies of glycyl-tRNA synthetases reveal that both the enzyme and tRNA undergo significant conformational changes during the aminoacylation process . For recombinant R. baltica glyQS, these conformational dynamics likely play critical roles in:
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Proper substrate recognition and binding
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Catalytic activation of glycine via adenylate formation
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Transfer of the glycyl moiety to tRNA
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Release of charged tRNA product
Research suggests a working model involving multiple conformations throughout the catalytic cycle. Alterations in these conformational changes could profoundly affect enzyme efficiency. Researchers should consider that proper subunit interactions are essential for full catalytic activity, as mutations disrupting these interactions have been shown to severely reduce adenylate synthesis activity (>100-fold reduction) in other glycyl-tRNA synthetases .
What are the kinetic parameters that define R. baltica glyQS efficiency?
When studying recombinant R. baltica glyQS, researchers should focus on determining the following kinetic parameters:
| Parameter | Expected Range | Experimental Approach |
|---|---|---|
| Km for glycine | 10-100 μM | Varying glycine concentration in aminoacylation assays |
| Km for ATP | 0.1-1.0 mM | Varying ATP concentration in aminoacylation assays |
| Km for tRNAGly | 0.5-5.0 μM | Varying tRNA concentration with saturating glycine and ATP |
| kcat | 1-10 s-1 | Time-course aminoacylation at saturating substrate concentrations |
| kcat/Km | 105-107 M-1s-1 | Calculated from independently determined kcat and Km values |
Measurement of these parameters under varying conditions (pH, temperature, salt concentration) would provide valuable insights into the catalytic properties of R. baltica glyQS and how they relate to the organism's marine environment and unique life cycle.
What expression systems are most effective for producing functional recombinant R. baltica glyQS?
When expressing recombinant R. baltica glyQS, researchers should consider several expression systems:
| Expression System | Advantages | Considerations |
|---|---|---|
| E. coli BL21(DE3) | High yield, simple protocol | May require codon optimization for R. baltica |
| E. coli Rosetta | Supplies rare tRNAs | Useful if codon bias issues exist |
| Insect cell systems | Better folding for complex proteins | Higher cost, longer expression time |
| Cell-free systems | Rapid, avoids toxicity issues | Lower yield, higher cost |
The marine origin of R. baltica suggests that salt concentration may be an important factor for proper folding and activity. R. baltica exhibits salt resistance in its native environment , so expression conditions should be optimized accordingly. Researchers should test expression under various IPTG concentrations (0.1-1.0 mM) and temperatures (18-37°C) to maximize the yield of soluble, active enzyme.
What purification strategies yield highest activity for recombinant R. baltica glyQS?
A multi-step purification approach is recommended:
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Affinity chromatography: His-tag or GST-tag purification as the initial capture step
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Ion exchange chromatography: To separate isoforms and remove nucleic acid contaminants
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Size exclusion chromatography: Final polishing step to ensure homogeneity
Critical buffer considerations include:
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Maintaining 5-10% glycerol to stabilize enzyme structure
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Including 1-5 mM DTT or β-mercaptoethanol to protect cysteine residues
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Testing salt concentration effects (100-500 mM NaCl)
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Optimizing pH range (typically 7.0-8.0)
Activity assays should be performed at each purification step to monitor retention of enzymatic function. Researchers should be aware that aminoacyl-tRNA synthetases can exhibit nonlinear dependence between activity and enzyme concentration due to potential disruption of subunit interactions, as observed with certain glycyl-tRNA synthetase mutants .
How can researchers design assays to accurately measure R. baltica glyQS activity?
Several complementary approaches can be used to assess recombinant R. baltica glyQS activity:
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ATP-PPi exchange assay: Measures the first step of the reaction (adenylate formation)
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Advantages: Simple, high-throughput
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Limitations: Does not assess complete tRNA charging
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Aminoacylation assay with radiolabeled glycine: Gold standard for full enzyme activity
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Advantages: Direct measurement of product formation
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Limitations: Requires radioisotope handling
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HPLC-based analysis of charged tRNA: Non-radioactive alternative
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Advantages: Safe, can assess multiple parameters
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Limitations: Lower sensitivity, requires specialized equipment
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Fluorescence-based assays: Using fluorescent tRNA derivatives
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Advantages: Real-time monitoring possible
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Limitations: Fluorescent modifications may alter enzyme kinetics
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Each assay should be performed under multiple conditions to determine the dependence of activity on temperature, pH, and ionic strength, with particular attention to salt concentration given R. baltica's marine origin .
How does R. baltica glyQS differ from other bacterial glycyl-tRNA synthetases?
Researchers investigating R. baltica glyQS should consider several potentially unique features compared to other bacterial glycyl-tRNA synthetases:
| Feature | Expected Distinctive Properties in R. baltica glyQS | Research Implications |
|---|---|---|
| Salt tolerance | Likely higher stability in elevated salt conditions | May require different buffer conditions for optimal activity |
| Substrate specificity | Potentially narrower or broader glycine analog tolerance | Important for inhibitor development and evolutionary studies |
| Quaternary structure | May have unique subunit arrangements | Critical for understanding assembly and activation mechanisms |
| Growth phase regulation | Expression varies with morphological changes | Time-dependent sampling important for native studies |
The genome analysis of R. baltica has revealed numerous unique features, including specialized sulfatases and C1-metabolism genes . This suggests that its aminoacyl-tRNA synthetases, including glyQS, may have evolved distinctive properties adapted to its unique ecological niche and cellular organization.
How can transcriptomic data inform R. baltica glyQS expression studies?
Transcriptomic studies of R. baltica throughout its growth cycle reveal significant differential gene expression patterns corresponding to its morphological transitions . For glyQS studies, this suggests:
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Expression levels may vary significantly depending on growth phase
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Sampling time should be carefully controlled when isolating native enzyme
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Regulatory elements may be growth phase-specific
Research has shown that R. baltica transitions through distinct morphological phases: swarmer and budding cells in early exponential phase, single and budding cells with rosettes in transition phase, and predominantly rosette formations in stationary phase . Each of these phases may have different translational demands and thus different requirements for aminoacyl-tRNA synthetase activity.
What evolutionary insights can be gained from studying R. baltica glyQS?
The Planctomycetes phylum, to which R. baltica belongs, exhibits intriguing evolutionary features. Studying its glyQS can provide insights into:
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Evolution of aminoacyl-tRNA synthetase specificity
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Adaptation of protein synthesis machinery to specialized cellular compartmentalization
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Divergence in substrate recognition mechanisms across bacterial phyla
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Co-evolution of tRNA and aminoacyl-tRNA synthetases
Comparative analysis of R. baltica glyQS with human glycyl-tRNA synthetase may be particularly informative. Human glycyl-tRNA synthetase mutations are associated with Charcot-Marie-Tooth disease, where mutant forms aberrantly bind to neuropilin 1 and Trk receptors . Understanding the structural and functional differences between these enzymes could provide insights into both evolutionary relationships and disease mechanisms.
How can researchers address insolubility issues with recombinant R. baltica glyQS?
Insolubility is a common challenge when expressing recombinant aminoacyl-tRNA synthetases. Researchers can implement several strategies:
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Lower expression temperature (16-20°C) to slow folding and reduce inclusion body formation
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Use solubility-enhancing fusion partners (SUMO, thioredoxin, MBP)
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Test different lysis buffer compositions:
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Increased salt concentration (250-500 mM)
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Addition of mild detergents (0.1% Triton X-100)
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Inclusion of osmolytes like glycerol (10-20%)
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Co-express with chaperone proteins (GroEL/GroES, DnaK/DnaJ)
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Express as separate domains and reconstitute activity
These approaches should be systematically tested and evaluated based on both yield and retained enzymatic activity, as conditions that improve solubility may not necessarily preserve function.
What are the critical factors affecting recombinant R. baltica glyQS stability?
Long-term stability of purified recombinant R. baltica glyQS is crucial for reliable experimental results. Key factors include:
| Stability Factor | Recommended Approach | Assessment Method |
|---|---|---|
| Storage temperature | Test -80°C, -20°C, 4°C | Activity retention over time |
| Cryoprotectants | 10-20% glycerol, 0.5-1M sucrose | Prevention of freeze-thaw damage |
| Reducing agents | Fresh DTT (1-5 mM) or TCEP (0.5-1 mM) | Protection of cysteine residues |
| Metal ions | Test EDTA vs. specific metal supplementation | Impact on structural integrity |
| Protein concentration | Higher concentration (>1 mg/ml) may improve stability | Concentration-dependent activity |
Since R. baltica is adapted to marine environments, stability in higher salt concentrations should be evaluated. Multiple freeze-thaw cycles should be avoided, and aliquoting of purified enzyme is recommended. Activity assays should be performed after various storage conditions to establish optimal protocols for maintaining enzyme function.
How can researchers validate the specificity of recombinant R. baltica glyQS?
Confirming the specificity of recombinant R. baltica glyQS is essential for reliable experimental outcomes. Researchers should implement multiple validation approaches:
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Substrate specificity testing:
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Assay aminoacylation with glycine analogs (alanine, serine)
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Determine charging efficiency with heterologous tRNAs
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Test activity with various ATP analogs
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Kinetic analysis:
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Compare Km values for cognate vs. non-cognate substrates
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Assess competitive inhibition patterns
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Structural validation:
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Circular dichroism to confirm proper folding
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Limited proteolysis to verify domain organization
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Thermal shift assays to assess structural stability
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Functional comparison:
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Complementation assays in bacterial expression systems
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In vitro translation efficiency measurements
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These approaches will help ensure that observed activities are attributable to specific, correctly folded recombinant R. baltica glyQS rather than contaminants or misfolded protein.
