Recombinant Pseudomonas putida Transcription elongation factor GreB (greB)

What is the basic structure and function of P. putida GreB?

GreB functions as a transcription elongation factor that promotes transcription by stimulating an endogenous, endonucleolytic transcript cleavage activity of RNA polymerase. The protein structure features an N-terminal coiled-coil domain that extends approximately 45 Å through a channel directly to the RNA polymerase active site . In Pseudomonas putida GB-1, GreB is encoded by gene PputGB1_1917 located on the chromosome at position 2157018-2157488 on the positive strand .

Key physicochemical properties of P. putida GreB include:

The slightly negative charge and hydrophilic nature align with its role as a soluble transcription factor that interacts with RNA polymerase .

How do conserved domains in GreB contribute to its function?

The functionality of GreB depends on several critical structural elements:

• The N-terminal coiled-coil domain extends through a channel to the RNA polymerase active site and is essential for the protein's function .

• Conserved acidic residues at the tip of the coiled-coil domain are critical for modifying the RNA polymerase active site to catalyze the transcript cleavage reaction. Mutational studies confirm these positions are crucial for Gre factor function .

• The C-terminal domain facilitates interactions with RNA polymerase, positioning the N-terminal domain correctly for transcript cleavage activity .

The functional mechanism involves GreB's ability to resolve backtracked transcription complexes by stimulating the RNA polymerase to cleave the nascent RNA transcript, allowing transcription to resume from the newly created 3' end .

What are the optimal expression systems for recombinant GreB in P. putida?

Based on research with other recombinant proteins in P. putida, several expression strategies have proven effective and could be adapted for GreB:

• Chromosomal integration via rRNA operons: Integration into ribosomal RNA (rRNA) operons has demonstrated exceptional efficiency for heterologous gene expression in P. putida. Studies show that all seven rrn operons can function as integration sites, with varying expression levels depending on the specific operon and proximity to the promoter .

• Transposon-based integration: Tn5 transposon-based chromosomal integration systems provide stable maintenance of heterologous genes without antibiotic selection pressure. This approach allows for screening of multiple integration loci to identify optimal expression sites .

• T7 RNA polymerase-dependent expression: This system has been successfully employed for heterologous gene expression in P. putida, though constitutive expression from native promoters (particularly rRNA promoters) has shown higher expression levels in some cases .

The following table summarizes key considerations for different expression approaches:

What purification strategies maintain GreB activity?

For optimal purification of active recombinant GreB, consider the following strategy:

• Expression construct design:

  • Include an affinity tag (His-tag or GST-tag) positioned to avoid interference with functional domains

  • Consider a cleavable tag system if the tag might affect activity

• Extraction conditions:

  • Gentle cell disruption methods to preserve protein structure

  • Buffer formulation with protease inhibitors and reducing agents

• Purification workflow:

  • Initial capture by affinity chromatography

  • Secondary purification via ion exchange chromatography (given GreB's pI of 5.37)

  • Final polishing by size exclusion chromatography

• Activity preservation:

  • Storage in buffer containing glycerol (10-20%) and reducing agents

  • Minimization of freeze-thaw cycles

  • Validation of activity after each purification step

How can researchers assess GreB-mediated transcript cleavage activity?

Several assays can evaluate the functional activity of recombinant GreB:

• In vitro transcript cleavage assay:

  • Components: Purified RNA polymerase, purified GreB, DNA template, nucleotides

  • Procedure: Form stalled or backtracked transcription complexes, add GreB, analyze RNA cleavage products

  • Detection: Radioactively or fluorescently labeled transcripts visualized by gel electrophoresis

• RNA polymerase binding assays:

  • Surface plasmon resonance or biolayer interferometry to determine binding kinetics

  • Pull-down assays to confirm physical interaction

• Structural visualization:

  • Cryo-electron microscopy has successfully visualized E. coli RNA polymerase-GreB complex at 15 Å resolution

  • This approach reveals the precise positioning of GreB relative to the RNA polymerase active site

• Functional complementation:

  • Assess the ability of recombinant GreB to rescue phenotypes in GreB-deficient strains

How can researchers study GreB's role in transcription elongation kinetics?

To investigate GreB's impact on transcription elongation rates and fidelity:

• Single-molecule approaches:

  • Optical tweezers or FRET-based methods to monitor individual transcription complexes

  • Real-time observation of transcript cleavage events and their effect on elongation

• Bulk biochemical assays:

  • Run-off transcription assays with defined templates

  • Measurement of transcript elongation rates with and without GreB

• Genome-wide approaches:

  • RNA-seq to identify genes whose expression is affected by GreB

  • NET-seq (nascent elongating transcript sequencing) to map RNA polymerase pause sites genome-wide

How can GreB be used as a tool for heterologous gene expression in P. putida?

Understanding GreB function can inform strategies for optimizing heterologous gene expression in P. putida:

• Co-expression strategies: Expressing additional GreB alongside heterologous genes could enhance transcription efficiency, particularly for genes with sequences prone to causing transcriptional pausing or backtracking .

• Integration site selection: Research has demonstrated that rRNA operons are particularly favorable sites for heterologous gene expression in P. putida. This knowledge can be applied when designing expression systems for recombinant proteins .

• Promoter engineering: Insights from GreB's interaction with RNA polymerase could inform the design of synthetic promoters with reduced transcriptional pausing.

Studies have shown that random chromosomal integration of heterologous genes in P. putida predominantly results in integration into rRNA operons when strong expression is selected for, indicating the importance of these genomic regions for efficient transcription .

What insights have comparative genomic analyses revealed about GreB in Pseudomonas species?

GreB appears to be widely conserved across Pseudomonas species, suggesting its fundamental importance in transcription regulation:

How does GreB contribute to bacterial stress responses?

While not directly addressed in the search results for P. putida specifically, research in related bacterial systems suggests GreB plays important roles in stress adaptation:

• Transcriptional fidelity: GreB helps resolve backtracked transcription complexes, which occur more frequently under stress conditions, thereby maintaining transcriptional output .

• Stress-specific gene expression: By facilitating efficient transcription elongation, GreB may be particularly important for expressing genes required for stress adaptation.

• Research approaches: To investigate GreB's role in P. putida stress responses, researchers could:

  • Compare growth of wild-type and greB knockout strains under various stress conditions

  • Analyze transcriptome changes in response to stress with and without functional GreB

  • Examine whether greB expression itself is regulated in response to specific stresses

What strategies are effective for creating and analyzing greB mutants in P. putida?

For systematic investigation of GreB function through mutational analysis:

• Site-directed mutagenesis approaches:

  • Target conserved acidic residues at the tip of the coiled-coil domain

  • Create truncation mutants to delineate domain functions

  • Introduce mutations that affect RNA polymerase binding without disrupting structural integrity

• Genomic modification strategies:

  • CRISPR-Cas9 for precise genomic editing

  • Homologous recombination-based approaches for gene replacement

  • Transposon mutagenesis for random insertional inactivation

• Phenotypic characterization:

  • Growth characteristics under various conditions

  • Sensitivity to antibiotics targeting transcription/translation

  • Transcriptome analysis to identify affected pathways

• Complementation studies:

  • Reintroduce wild-type greB to confirm phenotype causality

  • Test complementation with greB variants to identify critical functional residues

  • Perform cross-species complementation to assess functional conservation

How can researchers leverage P. putida's rRNA operons for GreB expression studies?

The finding that rRNA operons are favorable sites for heterologous gene expression in P. putida provides valuable insights for GreB expression studies:

• Targeted integration: Rather than random integration, researchers can specifically target the insertion of recombinant greB into different rRNA operons to compare expression levels .

• Promoter analysis: The distinctive characteristics of rRNA promoters that drive high expression levels can be analyzed and potentially incorporated into expression vectors .

• Comparative analysis: Studies have shown differential expression levels depending on which of the seven rrn operons contains the inserted gene and the distance between the integration site and the promoter .

• Experimental considerations: When inserting genes into rRNA operons, researchers should evaluate potential impacts on bacterial fitness, although studies indicate that P. putida strains with insertions in rRNA genes did not show apparent signs of cellular stress .

Research has demonstrated that chromosomal integration of heterologous genes via transposition in P. putida frequently results in integration into rRNA operons when screening for high expression, with all seven rrn operons capable of supporting expression but with varying efficiency .

What are the most promising future research directions involving P. putida GreB?

Several promising research avenues emerge from current understanding of GreB:

• Structural biology: High-resolution structures of P. putida GreB, particularly in complex with RNA polymerase, would provide valuable insights into species-specific adaptations.

• Synthetic biology applications: Exploring the potential of GreB co-expression to enhance heterologous protein production in P. putida-based bioprocesses.

• Stress adaptation: Investigating GreB's role in P. putida's renowned stress tolerance and metabolic versatility.

• Comparative transcriptomics: Genome-wide analyses comparing transcription dynamics in wild-type and greB-mutant strains under various conditions.

• Biotechnological applications: Leveraging insights about rRNA operons as integration sites for developing improved heterologous expression systems in P. putida.

The multifaceted roles of GreB in transcription regulation make it an intriguing subject for both fundamental research and biotechnological applications, particularly considering P. putida's importance as a host for synthetic biology and metabolic engineering .