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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

In vitro transcription assays can decipher the mechanisms of transcriptional regulation in Borreliella burgdorferi. This protocol describes the steps to purify B. burgdorferi RNA polymerase and perform in vitro transcription reactions. Experimental approaches using in vitro transcription assays require reliable purification and storage of active RNA polymerase.

Abstract

Borreliella burgdorferi is a bacterial pathogen with limited metabolic and genomic repertoires. B. burgdorferi transits extracellularly between vertebrates and ticks and dramatically remodels its transcriptional profile to survive in disparate environments during infection. A focus of B. burgdorferi studies is to clearly understand how the bacteria responds to its environment through transcriptional changes. In vitro transcription assays allow for the basic mechanisms of transcriptional regulation to be biochemically dissected. Here, we present a detailed protocol describing B. burgdorferi RNA polymerase purification and storage, sigma factor purification, DNA template generation, and in vitro transcription assays. The protocol describes the use of RNA polymerase purified from B. burgdorferi 5A4 RpoC-His (5A4-RpoC). 5A4-RpoC is a previously published strain harboring a 10XHis-tag on the rpoC gene encoding the largest subunit of the RNA polymerase. In vitro transcription assays consist of the RNA polymerase purified from strain 5A4-RpoC, a recombinant version of the housekeeping sigma factor RpoD, and a PCR-generated double-stranded DNA template. While the protein purification techniques and approaches to assembling in vitro transcription assays are conceptually well understood and relatively common, handling considerations for RNA polymerases often differ from organism to organism. The protocol presented here is designed for enzymatic studies on the B. burgdorferi RNA polymerase. The method can be adapted to test the role of transcription factors, promoters, and post-translational modifications on the activity of the RNA polymerase.

Introduction

Lyme disease and relapsing fever are caused by spirochete pathogens in the genera Borrelia and Borreliella1,2,3. Lyme disease is a prominent vector-borne disease in North America, and, consequently, Borreliella burgdorferi is a prominent model organism to study spirochete biology4,5. Investigations into the B. burgdorferi mechanisms of transcriptional regulation aim to better understand its adaptations to changes in the environment as it cycles between its tick vector and mammalian hosts6,7. Changes in pH, temperature, osmolarity, nutrient availability, short-chain fatty acids, organic acids, and dissolved oxygen and carbon dioxide levels modulate the expression of genes that are important for B. burgdorferi to survive in its arthropod vector and to infect animals8,9,10,11,12,13,14,15,16,17,18. Linking these responses to stimuli with regulatory mechanisms has been an important aspect of B. burgdorferi research19.

Transcription factors and sigma factors control the transcription of genes that carry out cellular processes. Lyme and relapsing fever spirochetes harbor a relatively sparse set of transcription factors and alternative sigma factors. Despite this, there are complex transcriptional changes directing B. burgdorferi responses to the environment20,21,22. The specific mechanisms driving transcriptional changes in B. burgdorferi in response to environmental changes remain unclear. In vitro transcription assays are powerful tools for employing a biochemical approach to assay the function and regulatory mechanisms of transcription factors and sigma factors23,24,25,26.

An in vitro transcription assay system using the B. burgdorferi RNA polymerase was recently established24. As bacteria often have unique cellular physiologies, RNA polymerases of different species and genera respond differently to enzyme purification, enzyme storage, and reaction buffer conditions27. B. burgdorferi is also genetically distant from the many bacterial species in which RNA polymerases have been studied20. Aspects of enzyme preparation such as lysis, wash, and elution buffer conditions, storage buffer, in vitro transcription reaction buffer, and the method of assay construction can all alter RNA polymerase activity. Herein, we provide a protocol for the purification of RNA polymerase and sigma factor RpoD, the production of linear double-stranded DNA template, and the construction of in vitro transcription assays to facilitate reproducibility between laboratories using this system. We detail an example reaction to demonstrate the linear range for RpoD-dependent transcription and discuss limitations and alternatives to this approach.

Protocol

1. Purification of the RNA polymerase and preparation of RNA polymerase stock

  1. Collect the cell pellet from 2-4 L of B. burgdorferi RpoC-His10X cultured in BSKII medium in a microaerophilic environment (34 °C, 5% CO2, 3% O2) to a density of 2-4 x 107 cells/mL using previously described cell collection protocols28,29. Perform centrifugation at 10,000 x g at 4 °C for 30 min in 500 mL centrifugal bottles and pour off the supernatant.
  2. Resuspend the cells in 30 mL of ice-cold HN buffer (10 mM HEPES, 10 mM NaCl, pH 8.0). Repeat the centrifugation step as described above using a 50 mL centrifuge tube and decant the supernatant.
    NOTE: Stopping point. Freeze the cell pellet at −80 °C to store cells for up to 2 weeks or immediately proceed to cell lysis.
  3. Maintain the cells, lysate, and purified proteins at 4 °C or on ice unless freezing. Keep RNA polymerase in a reducing environment by adding freshly prepared DTT at a concentration of 2 mM to every buffer used in this protocol and maintain pH 8.0.
  4. Prepare lysate from the B. burgdorferi cell pellet using a commercial bacterial lysis kit following the kit instructions. Resuspend the pellet in 10-15 mL of room temperature (RT) B-PER solution without protease inhibitor and allow lysis to proceed for 5 min on ice. The lysis volume depends on the cell pellet size, as described in the kit instructions.
  5. Add protease inhibitor cocktail to the lysis solution, followed by three rounds of sonication, as described in the representative results section.
    NOTE: Alternatively, lyse the cells in a French pressure cell as described previously24. Skip step 1.4. and step 1.5.
  6. Clarify the cell lysate using centrifugation and filtration using a 50 mL centrifuge tube. Fill the volume of the tube to at least 30 mL total volume by adding cobalt column loading buffer (Table 1) to the solution. Pellet the cell debris by centrifugation at 20,000 x g for 30 min.
  7. Filter the supernatant using a 0.45 µm syringe filter.
  8. Dilute the lysate in cobalt column loading buffer (Table 1) using a 1:10 final dilution ratio.
  9. Perform affinity chromatography on the clarified cell lysate supernatant using a cobalt or nickel-resin column following the manufacturer's instructions. See the representative results section for an example. Use the buffers listed in Table 1. Collect the flow-through, wash, and elution samples for analysis.
  10. Immediately exchange the RNA polymerase in the elution buffer solution into RNA polymerase storage buffer solution (Table 1) using a buffer exchange column following the manufacturer's instructions.
  11. Prepare the freezer stocks by concentrating the RNA polymerase using 10 kDa-cutoff centrifugal filter units to a concentration of 0.2-0.4 mg/mL determined by spectrophotometry (A280nm without extinction coefficient adjustment [1 abs at 1 cm = 1 mg·mL−1]).
  12. Aliquot RNA polymerase freezer stocks in 20-50 µL volumes in PCR tubes and store the RNA polymerase at −80 °C.
  13. Determine the quality of the affinity chromatography by SDS-PAGE and measure the total protein yield by spectrophotometry or BCA assay. Run the samples on a 7.5% polyacrylamide gel at 120 V for 50 min for good resolution by SDS-PAGE.

2. Purification of recombinant RpoD and preparation of RpoD stock

  1. Culture and induce the expression of Maltose Binding Protein-tagged recombinant B. burgdorferi RpoD (MBP-RpoD) in BL21::MBP-RpoDBb, as described by the protein fusion and purification system instruction manual listed in the Table of Materials, and collect the cells by centrifugation.
    NOTE: Stopping point. Freeze the cell pellets at −80 °C to store cells for up to 2 weeks or immediately proceed to cell lysis.
  2. Perform lysis using a commercial bacterial cell lysis kit or French pressure cell. See the example lysis buffer in Table 1.
  3. Clarify the cell lysate using centrifugation and filtration. Pellet the cell debris by centrifugation at 20,000 x g for 30 min. Filter the supernatant using a 0.45 µm filter.
  4. Extract MBP-RpoD using a protein extraction protocol for the expression system. See the representative results section for example implementation details and results.
  5. Determine the quality of the affinity chromatography process and elution by SDS-PAGE and measure the protein yield by spectrophotometry (A280nm without extinction coefficient adjustment [1 abs at 1 cm = 1 mg·mL−1]). Use 10% polyacrylamide gel at 200 V for 30 min for separation in SDS-PAGE.
  6. Concentrate MBP-RpoD using a 10 kDa-cutoff centrifugal filter to a concentration of >2 mg/mL determined by spectrophotometry.
  7. Cleave MBP from RpoD at the Factor Xa cleavage site. Add CaCl2 to the affinity chromatography elution fractions containing MBP-RpoD at a concentration of 2 mM. Add 200 µg of Factor Xa Protease for every 30 mg of purified protein. Incubate overnight at RT.
  8. Confirm complete cleavage of MBP from RpoD by SDS-PAGE. Use 10% polyacrylamide gel at 200 V for 30 min for separation in SDS-PAGE.
  9. Dilute the MBP and RpoD-containing solution at a ratio of 1:10 with a heparin-binding buffer.
  10. Separate MBP and RpoD on the heparin column by FPLC. See Table 2 for FPLC settings.
  11. Determine the quality of the affinity chromatography products by SDS-PAGE and measure the protein yield by spectrophotometry. Use 10% polyacrylamide gel at 200 V for 30 min for separation in SDS-PAGE.
  12. Concentrate the elution fractions containing RpoD using a 10 kDa-cutoff centrifugal filter to a final volume of 2-3 mL.
  13. Buffer exchange the concentrated RpoD elution fractions to storage buffer using a gel filtration column.
  14. Concentrate the RpoD stock using a 10 kDa-cutoff centrifugal filter to a concentration of 0.2-0.8 mg/mL as determined by spectrophotometry.
  15. Aliquot RpoD stock at 20-50 µL volumes in PCR tubes and store at −80 °C.

3. Preparation of DNA template stock

  1. PCR amplify the 500 bp region surrounding an RpoD driven promoter site from B. burgdorferi genomic DNA using a 200 µL reaction (Table 3).
  2. Purify the PCR products using a commercial PCR purification kit. Elute the DNA in molecular biology grade H2O.
  3. Adjust the molar concentration of the PCR-generated DNA templates to 100 nM by dilution in molecular biology grade H2O.

4. Perform in vitro transcription with the incorporation of radiolabeled nucleotides

  1. Prepare an in vitro transcription experimental plan. Determine the RNA polymerase, RpoD, and DNA template concentrations. Determine the aliquot volumes and pipetting orders for the reaction tubes. See Table 4 and Figure 1 for an example.
    NOTE: Include controls in the experimental plan, such as reactions containing no RNA polymerase, alternative polymerase, no template, or alternative templates.
  2. Calculate the volume of α-32P-ATP required for the experiment. Incorporate this into the experimental plan. Typically, include 2 µCi of activity per in vitro transcription reaction for phosphor screen detection.
  3. Prepare the work surfaces, including the radiation bench, to reduce the radiation contamination risk.
  4. Thaw fresh aliquots of RNA polymerase, Rpo, 5x buffer NTP, and template stock solutions on ice. Mix all the thawed frozen stocks thoroughly prior to pipetting.
  5. Prepare a master reaction mixture and a separate NTP mixture for radiolabeled nucleotides according to the experimental plan.
  6. Dispense the control reactions and experimental sets into PCR tubes in preparation of adding radiolabel in the reaction. Gently dispense H2O, reaction master mix, RNA polymerase, and RpoD into designated PCR tubes and mix by pipetting.
  7. Transfer the prepared materials to a radiation bench.
  8. Add α-32P-ATP to NTP and mix by gentle pipetting.
    CAUTION: While working with radioactive material, use appropriate protective equipment and handling procedures for radioactive isotopes.
  9. Add NTP to the tubes containing the in vitro transcription reaction mixtures from step 4.6.
  10. Start the in vitro transcription reaction with the addition of a DNA template. Mix the reaction volume by gently pipetting.
  11. Incubate tubes at 37 °C for 5 min in a thermocycler or heat block.
  12. Remove the reactions from the thermocycler or heat block and add the 2x RNA loading dye containing 50% formamide to an equal reaction volume to stop in vitro transcription reactions.
  13. Denature the enzymes by incubating reactions at 65 °C for 5 min in a thermocycler or heat block.
  14. Separate RNA in the in vitro transcription reaction mixture by gel electrophoresis using 10%-15% TBE urea polyacrylamide gels at 180 V for 30-45 min.
    NOTE: Depending on the gel electrophoresis conditions, the buffer solution may be contaminated with radiolabeled nucleotides or the gel may contain most or all of the radiolabeled nucleotides. Plan for proper radioactivity storage or disposal.
  15. Image the radiolabeled RNA using phosphoscreen after removing any portion of the gel containing unincorporated radiolabeled ATP. Expose the gel to the phosphoscreen overnight (16 h) and image using a phosphoscreen imager (100 µm resolution, 700 V PMT tube voltage).
  16. Analyze the data using densitometry methods24.

Results

In an in vitro transcription reaction in which the limiting step of the reaction is sigma factor-mediated transcription initiation, the transcription activity should increase linearly with the amount of sigma factor. We present the preparation of in vitro transcription experiments testing a range of RpoD concentrations along with two concentrations of RNA polymerase to observe the resulting varying signal from radiolabeled nucleotide incorporation into RNA products. Representative results of the prepara...

Discussion

In vitro transcription assays constructed using the presented protocol were recently used to study the role of a transcription factor in B. burgdorferi and can be applied to build similar experiments using other transcription factors, sigma factors, and molecules23. Once active RNA polymerase from B. burgdorferi has been obtained and its activity detected, components and conditions within the in vitro transcription assays can be modified. The assay is highly fle...

Disclosures

The authors declare no conflicts of interest.

Acknowledgements

This work was supported by the Health Sciences Strategic Investment Fund Faculty Development Grant of Creighton University. The B. burgdorferi RpoC-His10X strain was kindly provided by Dr. D. Scott Samuels of the University of Montana. The E. coli strain harboring the pMAL-C5X plasmid encoding a maltose-binding protein-tagged rpoD allele was kindly provided by Dr. Frank Gherardini of Rocky Mountain Laboratories, NIAID, NIH.

Materials

NameCompanyCatalog NumberComments
0.45 micron syringe filterThermo Scientific726-2545Step 1.7 and 2.3
50 mL conical tubesMidSciC50BStep 1.3, and subsequent steps
50 mL high-speed centrifuge tubesThermo Scientific3119-0050PKStep 1.2
500 mL Centrifuge bottlesThermo Scientific3120-9500PKStep 1.1
B-PER and instruction manualThermo Scientific78248Step 1.4 and 2.2
Calcium chlorideFisher Scientific10035-04-8Step 2.6
Centrifugal filters 10 Kd cutoffMillipore SigmaUFC8010Step 1.11 and 2.11
Cobalt resin and instruction manualThermo Scientific89969Step 1.9
DithiothreitolAcros Organics426380500Step 1.4 and subsequent steps
Dnase (Nuclease)Millipore Sigma70746Step 1.4 and 2.2
Factor Xa Protease Haematologic TechnologiesHCXA-0060Step 2.6
GE Typhoon 5 PhosphoimagerGE lifesciencesMultipleStep 4.15
Gel ImagerBio-RadMutipleStep 1.13 and subsequent protien quality check steps
H2O for in vitro transcriptionFisher Scientific7732-18-5Step 3.2 and 3.3
high fidelity PCR kitNew England BiolabsM0530SStep 3.1
High-speed centrifugeEppendorfStep 1.1, and subsequent steps
HiTrap Heparin HP 5 x 1 mLCytiva Life Sciences17040601Step 2.8
ImidazoleSigma-Aldrich56750-100GStep 1.9
LysozymeThermo Scientific90082Step 1.4 and 2.2
Magnesium chloride Fisher ScientificS25401Step 4.1
Manganese chlorideFisher ScientificS25418Step 4.1
Mini protean tetra cellBio-RadMutipleStep 1.13 and subsequent protien quality check steps
NP-40Thermo Scientific85124Step 4.1
NTP mixtureThermo ScientificR0481Step 4.1
PCR purification kitQiagen28506Step 3.2
PCR tubesMidSciPR-PCR28ACFStep 1.12
PD-10 Sephadex buffer exchange column and instruction manualCytiva17085101Step 1.10 and 2.10 (gel filtration column)
pMAL Protein Fusion
and Purification System Instruction manual		New England BiolabsE8200SStep 2.1
Polyacrylamide gels AnyKDBio-Rad456-8125Step 1.13 and subsequent protien quality check steps
Potassium glutamateSigma-AldrichG1251Step 4.1
Protease inhibitorThermo Scientific78425Step 1.4 and 2.2
Radiolabeled ATPPerkin ElmerBLU503HStep 4.2
RNA Loading Dye, (2x)New England BiolabsB0363SStep 4.13
Rnase inhibitorThermo ScientificEO0381Step 4.1
SpectrophotometerBiotekMutipleStep 1.13 and subsequent protien quality check steps
TBE-Urea gels 10 percentBio-Rad4566033Step 4.14

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