Recombinant Rhizobium sp. Uncharacterized HTH-type transcriptional regulator y4fK (NGR_a03710)
Description
Introduction to Recombinant Rhizobium sp. Uncharacterized HTH-type Transcriptional Regulator y4fK (NGR_a03710)
The Recombinant Rhizobium sp. Uncharacterized HTH-type transcriptional regulator y4fK, also known as NGR_a03710, is a protein derived from the Rhizobium species. This protein is part of the helix-turn-helix (HTH) family of transcriptional regulators, which play crucial roles in bacterial gene expression by binding to specific DNA sequences. Despite its designation as "uncharacterized," this protein is of interest due to its potential involvement in regulating various cellular processes within Rhizobium species.
Structure and Expression
The full-length recombinant protein consists of 427 amino acids and is expressed in Escherichia coli with an N-terminal His tag for purification purposes . The protein is available in a lyophilized powder form with a purity of greater than 90% as determined by SDS-PAGE .
| Specification | Description |
|---|---|
| Protein Length | Full Length (1-427aa) |
| Source | E. coli |
| Tag | His |
| Purity | >90% by SDS-PAGE |
| Storage | -20°C/-80°C |
Function and Potential Applications
While specific functions of the y4fK transcriptional regulator are not well-documented, HTH-type regulators generally control gene expression in response to environmental cues, which could include symbiotic interactions with plant hosts. Rhizobium species are known for their symbiotic relationships with legumes, where they fix nitrogen, a process critical for plant growth. Understanding the role of y4fK could provide insights into optimizing these symbiotic interactions.
Research Findings and Availability
Research on this specific protein is limited, but it is commercially available from several suppliers, including MyBioSource.com, where it is priced at $1,895.00 . The partial form of this protein is also available for comparative studies .
Future Directions
Future research should focus on elucidating the specific regulatory roles of y4fK in Rhizobium species, particularly in the context of symbiotic interactions with legumes. This could involve transcriptomic analyses to identify target genes and functional assays to assess its impact on symbiosis and nitrogen fixation.
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your format preference during order placement for customized preparation.
Note: All proteins are shipped with standard blue ice packs. Dry ice shipping requires prior arrangement and incurs additional charges.
The tag type is finalized during production. If you require a specific tag, please inform us, and we will prioritize its development.
Target Background
KEGG: rhi:NGR_a03710
Q&A
What is the HTH-type transcriptional regulator y4fK (NGR_a03710) in Rhizobium sp.?
The y4fK (NGR_a03710) is an uncharacterized helix-turn-helix (HTH) type transcriptional regulator from Rhizobium sp., specifically identified in Sinorhizobium fredii. It belongs to a class of DNA-binding proteins that regulate gene expression by binding to specific DNA sequences. The full-length protein consists of 427 amino acids and has a UniProt ID of P55449. As an HTH-type regulator, it likely contains a characteristic DNA-binding domain that adopts a helix-turn-helix conformation, enabling sequence-specific interactions with target DNA . While its exact function remains uncharacterized, comparative analysis with other HTH-type transcriptional regulators suggests it may play roles in regulating genes involved in symbiotic relationships, nitrogen fixation, or other metabolic processes in Rhizobium species.
How should recombinant y4fK protein be stored and handled in laboratory settings?
The optimal storage and handling of recombinant y4fK protein requires specific conditions to maintain stability and functionality. Based on established protocols for similar recombinant proteins, the following guidelines should be implemented:
| Storage Parameter | Recommended Condition | Notes |
|---|---|---|
| Long-term storage | -20°C to -80°C | Aliquoting is necessary to avoid repeated freeze-thaw cycles |
| Working storage | 4°C | For up to one week |
| Buffer composition | Tris/PBS-based buffer with 6% Trehalose, pH 8.0 | Maintains protein stability |
| Reconstitution | Deionized sterile water (0.1-1.0 mg/mL) | Centrifuge vial briefly before opening |
| Cryoprotectant | 5-50% glycerol (50% recommended) | For long-term storage aliquots |
| Freeze-thaw cycles | Avoid repeated cycles | Causes protein degradation |
The recombinant y4fK protein is typically supplied as a lyophilized powder with greater than 90% purity as determined by SDS-PAGE . Before reconstitution, the vial should be briefly centrifuged to bring contents to the bottom. After reconstitution, researchers should prepare multiple small aliquots to prevent repeated freeze-thaw cycles, which can significantly compromise protein integrity and functionality.
What expression systems are recommended for producing recombinant y4fK protein?
The expression of recombinant y4fK protein has been successfully achieved using Escherichia coli as the heterologous expression host. This system offers several advantages for the production of this Rhizobium sp. transcriptional regulator:
| Expression System Parameter | Implementation for y4fK | Advantages |
|---|---|---|
| Host organism | E. coli | Rapid growth, high yield, established protocols |
| Vector type | Expression vector with N-terminal His-tag | Facilitates purification via affinity chromatography |
| Protein length | Full length (1-427 amino acids) | Preserves complete functional domains |
| Purification approach | His-tag affinity chromatography | Yields >90% purity as determined by SDS-PAGE |
| Expression verification | SDS-PAGE and Western blotting | Confirms correct size and identity |
While E. coli is the documented expression system for this protein , researchers investigating specific protein-protein or protein-DNA interactions might consider alternative expression systems. For studies requiring post-translational modifications, yeast expression systems (S. cerevisiae or P. pastoris) might be more appropriate. For structural studies requiring proper folding of membrane-associated domains (as suggested by the amino acid sequence), insect cell or mammalian cell expression systems could be considered, though these would require optimization.
What structural domains are predicted in the y4fK transcriptional regulator?
Based on sequence analysis and comparison with characterized HTH-type transcriptional regulators, the y4fK protein likely contains several functional domains:
| Domain | Approximate Position | Predicted Function | Confidence Level |
|---|---|---|---|
| N-terminal region | 1-50 | Potential membrane association | Moderate |
| HTH motif | ~100-160 | DNA binding | High |
| Central domain | ~160-300 | Ligand binding/dimerization | Moderate |
| C-terminal domain | ~300-427 | Effector binding/oligomerization | Moderate |
The amino acid sequence contains hydrophobic regions in the N-terminus (LVLCLLVAGTIAALIAVLLYPP), suggesting possible membrane association . The presence of the characteristic HTH motif is expected based on its classification, though the exact boundaries would require experimental verification through structural studies or limited proteolysis. Many HTH-type transcriptional regulators function as dimers or higher-order oligomers, with distinct domains for DNA binding, effector molecule sensing, and dimerization. Detailed structural studies, including X-ray crystallography or cryo-electron microscopy, would be necessary to definitively map these domains and understand their functional interplay.
How does the His-tag affect the function of recombinant y4fK protein in experimental assays?
The addition of a His-tag to the recombinant y4fK protein is primarily intended for purification purposes but may impact protein function in various experimental contexts:
| Assay Type | Potential Impact of His-tag | Mitigation Strategy |
|---|---|---|
| DNA-binding assays | May interfere with binding if near DNA-binding domain | Consider C-terminal tag or tag removal |
| Protein-protein interaction studies | May create artificial interactions or block natural ones | Validate with both tagged and untagged versions |
| Structural studies | May introduce flexibility affecting crystallization | Remove tag via protease cleavage site |
| In vivo functional assays | May affect cellular localization or assembly | Compare with native protein expression |
| Enzymatic activity assays | Generally minimal impact if not near active site | Include appropriate controls |
What DNA motifs are potentially recognized by the y4fK transcriptional regulator?
While the specific DNA motifs recognized by y4fK have not been definitively characterized, several approaches can be employed to identify these sequences:
| Approach | Methodology | Expected Outcome | Limitations |
|---|---|---|---|
| ChIP-seq | Chromatin immunoprecipitation followed by sequencing | Genome-wide binding sites | Requires specific antibody or tagged version |
| ChIP-exo | Higher resolution variant of ChIP-seq | Precise binding site boundaries | Technical complexity |
| SELEX | Systematic evolution of ligands by exponential enrichment | Consensus binding motif | In vitro conditions may not reflect in vivo binding |
| DNA footprinting | Protection of DNA from enzymatic or chemical cleavage | Direct physical binding site | Limited throughput |
| Bacterial one-hybrid | Screenable reporter activation by DNA-protein interaction | Novel binding sequences | May yield false positives |
Drawing from studies of other HTH-type transcriptional regulators, binding sites often consist of palindromic or semi-palindromic sequences of 12-20 base pairs . The binding strength to specific DNA sequences is often correlated with the regulatory impact, as demonstrated in studies of other transcription factors where peak strength in ChIP-exo experiments correlates with expression changes when the site is targeted with dCas9-VPR . For y4fK specifically, initial screening could focus on promoter regions of genes co-regulated with NGR_a03710 or genes involved in similar biological processes in Rhizobium sp.
How can CRISPR-based techniques be used to study the function of y4fK in Rhizobium sp.?
CRISPR-based technologies offer powerful approaches for functional characterization of y4fK:
When designing gRNAs for CRISPR-based approaches, rational design principles should be employed. Recent research has shown that considering transcription factor binding sites when designing gRNAs can significantly impact success. For instance, targeting dCas9-VPR to non-motif regions outside of transcription factor binding sites tends to increase gene expression, while targeting directly to binding motifs can decrease expression due to competition with the native transcription factor . For studying y4fK itself, designing gRNAs that target different regions of the gene could reveal domain-specific functions. Additionally, targeting the binding sites of y4fK (once identified) could help elucidate its regulatory network.
What experimental approaches would be most effective for identifying the regulon of y4fK?
Identifying the complete set of genes regulated by y4fK (its regulon) requires integrated approaches:
| Approach | Methodology | Data Output | Integration Strategy |
|---|---|---|---|
| RNA-seq after y4fK deletion/overexpression | Differential gene expression analysis | Genes with altered expression | Primary regulon identification |
| ChIP-seq/ChIP-exo | Genome-wide binding site identification | Direct binding locations | Distinguishes direct vs. indirect regulation |
| Proteomics | Mass spectrometry of wild-type vs. mutant | Protein-level changes | Post-transcriptional effects |
| Metabolomics | Metabolite profiling | Metabolic consequences | Downstream effects of regulation |
| Protein-protein interaction studies | Co-IP, Y2H, or BioID | Interaction partners | Co-regulators and regulatory complexes |
| Phenotypic microarrays | Growth under various conditions | Condition-specific functions | Physiological relevance |
A particularly effective approach would be to first identify direct binding sites using ChIP-seq or ChIP-exo, which has been successfully applied to other transcriptional regulators . This could be followed by RNA-seq comparing wild-type and y4fK knockout strains grown under various conditions to determine which binding events result in functional regulation. Integration of these datasets would reveal both direct and indirect regulatory effects. For example, studies of other transcription factors have shown that only 10-20% of binding events may lead to expression changes, highlighting the importance of complementary approaches . Additionally, metabolomic and phenotypic analyses could link the regulon to specific physiological processes in Rhizobium sp.
How does y4fK compare structurally to other characterized HTH-type transcriptional regulators?
While the specific structure of y4fK has not been determined, comparative analysis with other HTH-type transcriptional regulators provides insights:
| HTH Regulator Family | Key Structural Features | Functional Implications | Similarity to y4fK |
|---|---|---|---|
| TetR family | N-terminal HTH domain, C-terminal ligand-binding domain | Responds to small molecules, often involved in antibiotic resistance | Moderate sequence similarity in HTH region |
| LysR family | N-terminal HTH domain, effector-binding domain | Responds to co-inducer molecules | Low sequence similarity |
| AraC/XylS family | Two HTH motifs, C-terminal dimerization domain | Carbon source utilization regulation | Low sequence similarity |
| YxaF (B. subtilis) | 191 aa, HTH domain identified | Function not fully characterized | Low-moderate similarity in HTH region |
What role might y4fK play in Rhizobium-legume symbiosis?
Given that y4fK is found in Rhizobium sp., which is known for forming symbiotic relationships with legumes, its potential role in symbiosis warrants investigation:
| Aspect of Symbiosis | Potential Role of y4fK | Experimental Approach | Expected Outcome |
|---|---|---|---|
| Nodulation | Regulation of nod genes | y4fK knockout and symbiosis assays | Altered nodulation phenotype |
| Nitrogen fixation | Control of nif/fix gene expression | Acetylene reduction assays with mutants | Changed nitrogen fixation efficiency |
| Host specificity | Regulation of host-specific factors | Cross-inoculation experiments | Altered host range |
| Stress response | Adaptation to plant microenvironment | Stress exposure transcriptomics | Differential expression during stress |
| Quorum sensing | Integration with population density signals | Autoinducer binding assays | Interaction with quorum molecules |
To investigate these potential roles, researchers could perform symbiotically relevant phenotypic assays comparing wild-type and y4fK mutant strains. These could include nodulation assays (counting nodules, measuring nodule size and leghemoglobin content), nitrogen fixation measurements, competitive nodulation assays, and plant growth promotion measurements. Transcriptomic analyses of bacteroids (symbiotic form) versus free-living bacteria could reveal condition-specific regulation by y4fK. Additionally, localization studies using fluorescently tagged y4fK could determine if its expression or cellular distribution changes during different stages of symbiosis.
