Essential Components of Borreliella (Borrelia) burgdorferi In Vitro Transcription Assays

William K. Boyle; Hannah N. Sorensen; Travis J. Bourret

doi:10.3791/64134

JoVE Journal > Immunology and Infection

Immunologie und Infektion

Essential Components of Borreliella (Borrelia) burgdorferi In Vitro Transcription Assays

Published: July 22, 2022

doi:

10.3791/64134

William K. Boyle¹, Hannah N. Sorensen¹, Travis J. Bourret¹

¹School of Medicine,Creighton University

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

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.
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.
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.
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.
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.
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.
Filter the supernatant using a 0.45 µm syringe filter.
Dilute the lysate in cobalt column loading buffer (Table 1) using a 1:10 final dilution ratio.
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.
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.
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 (A_280nm without extinction coefficient adjustment [1 abs at 1 cm = 1 mg·mL⁻¹]).
Aliquot RNA polymerase freezer stocks in 20-50 µL volumes in PCR tubes and store the RNA polymerase at −80 °C.
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

Culture and induce the expression of Maltose Binding Protein-tagged recombinant B. burgdorferi RpoD (MBP-RpoD) in BL21::MBP-RpoD_Bb, 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.
Perform lysis using a commercial bacterial cell lysis kit or French pressure cell. See the example lysis buffer in Table 1.
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.
Extract MBP-RpoD using a protein extraction protocol for the expression system. See the representative results section for example implementation details and results.
Determine the quality of the affinity chromatography process and elution by SDS-PAGE and measure the protein yield by spectrophotometry (A_280nm 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.
Concentrate MBP-RpoD using a 10 kDa-cutoff centrifugal filter to a concentration of >2 mg/mL determined by spectrophotometry.
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.
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.
Dilute the MBP and RpoD-containing solution at a ratio of 1:10 with a heparin-binding buffer.
Separate MBP and RpoD on the heparin column by FPLC. See Table 2 for FPLC settings.
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.
Concentrate the elution fractions containing RpoD using a 10 kDa-cutoff centrifugal filter to a final volume of 2-3 mL.
Buffer exchange the concentrated RpoD elution fractions to storage buffer using a gel filtration column.
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.
Aliquot RpoD stock at 20-50 µL volumes in PCR tubes and store at −80 °C.

3. Preparation of DNA template stock

PCR amplify the 500 bp region surrounding an RpoD driven promoter site from B. burgdorferi genomic DNA using a 200 µL reaction (Table 3).
Purify the PCR products using a commercial PCR purification kit. Elute the DNA in molecular biology grade H₂O.
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

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.
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.
Prepare the work surfaces, including the radiation bench, to reduce the radiation contamination risk.
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.
Prepare a master reaction mixture and a separate NTP mixture for radiolabeled nucleotides according to the experimental plan.
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.
Transfer the prepared materials to a radiation bench.
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.
Add NTP to the tubes containing the in vitro transcription reaction mixtures from step 4.6.
Start the in vitro transcription reaction with the addition of a DNA template. Mix the reaction volume by gently pipetting.
Incubate tubes at 37 °C for 5 min in a thermocycler or heat block.
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.
Denature the enzymes by incubating reactions at 65 °C for 5 min in a thermocycler or heat block.
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.
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).
Analyze the data using densitometry methods²⁴.

Representative 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 preparation of RNA polymerase, RpoD, and double-stranded DNA template stocks are shown. The representative in vitro transcription reactions serve as an example of RNA polymerase activity levels acceptable for analysis.

For the purification of RNA polymerase, a 4 L culture of B. burgdorferi RpoC-His10X was grown at 34 ˚C under microaerophilic conditions (5% CO₂, 3% O₂). The cultures were closely monitored for spirochete density using dark-field microscopy and collected prior to reaching densities higher than 4 x 10⁷ spirochetes/mL. The cells were resuspended in B-PER mixture for lysis (Table 1). The cell pellet was homogenized by pipetting, followed by 5 min incubation at RT, and subsequent rounds of sonication performed for 3 s in triplicate. Clarified lysates were diluted with cobalt column loading buffer and 5 mL of cobalt resin to a total volume of 180 mL. RpoC-His10X was allowed to bind to the cobalt resin by nutating the mixture for 1 h at 4 °C. The mixture was transferred to a gravity flow column and washed five times with 40 mL of column wash buffer. Bound protein was released by passing 5 mL of elution buffer through the column, and elution was repeated seven times. Samples of the flow-through, washes, and elution fractions were separated by SDS-PAGE and imaged by stain incorporation (Figure 1). The RNA polymerase core complex is composed of RpoC, RpoB, and RpoA, sized 154 kDa, 129 kDa, and 38.5 kDa, respectively. The representative result displayed in Figure 1 shows three prominent bands at the sizes of the three peptides composing the RNA polymerase complex. The flow-through from affinity chromatography contained 0.5-1 mg/mL of protein with a 260/280 ratio between 1.0 and 1.7, indicating a higher concentration of nucleic acids. The RNA polymerase protein yield following the concentration of the protein mixture as determined by spectrophotometry was 0.3 mg/mL.

To prepare RpoD protein stocks, recombinant RpoD with a C-terminal Maltose Binding Protein tag was expressed from a pMAL-C5X plasmid in BL21 (DE3) pLysS E. coli. 2 L of E. coli was grown at 32 °C to an OD600_nm of 0.5. The culture was then treated with 0.3 mM IPTG to induce the expression of RpoD for 1 h. The cells were pelleted by centrifugation and lysed in B-PER. The resulting 200 mL of diluted cell lysate was filtered through 10 mL of amylose resin. The resin was washed with 40 mL of binding buffer eight times to remove residual DNA and peptides. MBP-tagged RpoD was eluted with 5 mL of elution buffer into the resin column five times. The flow-through from the binding and washing steps and the elution fractions were analyzed by SDS-PAGE (Figure 2A). The major component in the elution fraction is a peptide of ~115 kDa, matching the size of the MBP-tagged recombinant B. burgdorferi RpoD. The elution fractions were combined, the CaCl₂ concentration was adjusted to 2 mM, and 100 µg of FactorXa protease was added to 40 mg of purified protein. Following overnight digestion at room temperature, the reaction was analyzed by SDS-PAGE (Figure 2B). Following cleavage with Factor Xa protease, the two major products in the mixture were RpoD (73.6 kDa) and MBP (45 kDa). The mixture of RpoD and MBP proteins was separated using heparin affinity chromatography. The mixture was diluted at a 1:10 ratio in heparin-binding buffer and loaded into a heparin-bound resin column in an FPLC. RpoD was eluted with an increasing gradient of NaCl to a concentration of 1 M. When the fractions were analyzed by SDS-PAGE, a major band of 73.6 kDa size was apparent in the elution fraction in the range of 400-600 mM NaCl (Figure 2C). Some degraded RpoD fragments were co-purified. RpoD-containing fractions were combined, buffer exchanged into RpoD storage buffer, and then concentrated using a 10 kDa-cutoff centrifugal filter. The final RpoD concentration was 0.67 mg/mL (9.1 mM) as determined by spectrophotometry.

A double-stranded DNA template encompassing the promoter site of B. burgdorferi flgB was generated by PCR. The flgB promoter was amplified by primers that corresponded to 250 bp upstream and downstream of the promoter (Table 6) using a 200 µL reaction containing 30 ng of B. burgdorferi DNA²⁴^,³⁰. The PCR product was analyzed by gel electrophoresis in a 1% agarose gel (Figure 3). The PCR product was roughly 500 bp in size with apparent homogeneity. Importantly, salts and cell component contamination can contribute to the variability of in vitro transcription reaction outcomes; we ensure that the DNA template is thoroughly desalted on the PCR purification column.

An experimental scheme in which transcription is initiated by the addition of template DNA can test the rate of promoter recognition and transcription initiation. In our representative experiments, a dilution series of RpoD protein was prepared by twofold dilutions in water ranging from 16 nM-1 µM. A master mix was created containing RNA polymerase in a reaction buffer containing divalent cations required for activity (Table 4 and Table 5). The RpoD and master mix were aliquoted together on ice. The master mix containing RNA polymerase and RpoD was allowed to incubate while other steps of the in vitro transcription were prepared. We incorporated three control reactions: a positive control reaction prepared separately from the master mix, a negative control reaction containing no template DNA to ensure the in vitro transcription signal was template dependent, and a negative control reaction without RNA polymerase to ensure the signal was RNA polymerase dependent. Radiolabeled nucleotides were added to the reaction by mixing 5 μL of (α-³²P-ATP) to 20 μL of 10x NTP stock mixture to create enough aliquot for 20 reactions to compensate for unrecoverable volumes. Finally, template DNA encoding the flgB promoter was introduced to the reaction tubes by pipetting following the previously planned pipetting order (Figure 4).

The signal from the RNA fragments generated by in vitro transcription was detected by phosphor screen following a 16 h exposure period of the TBE-urea gel containing RNA separated by gel electrophoresis. An experiment containing 50 nM of RNA polymerase and RpoD in the 500 nM to 16 nM range generated RNA products of 250 bp (Figure 5). For each twofold dilution of RpoD concentration, there was a lower level of accumulated RNA product. There was a series of lower bp products that appeared below the prominent band, indicating incomplete RNA fragments due to transcription halting or fragmented DNA template. RNA products were absent from the no template control, indicating that there were no exogenous DNA fragments contributing to the signal in this assay. No signal was detected in the no RNA polymerase control, indicating the B. burgdorferi RNA polymerase was the only polymerase contributing to the production of RNA in this assay.

In a second experiment, we used a lower concentration of 25 nM RNA polymerase (Figure 6). Fewer RNA products were detected across the range of RpoD concentrations tested compared to Figure 5, and the background signal intensity was higher. This is problematic for downstream analysis by densitometry. When the phosphor image was analyzed by densitometry (Figure 7), the densitometry signal obtained from the phosphoscreen indicated a linear relationship between the amount of RpoD present in the reaction in both experiments. However, in the experiment with high background signal, the reaction containing 25 nM RNA polymerase and less than 100 nM of RpoD was unreliable for relative transcription level comparisons.

Figure 1: Separation of His-tagged B. burgdorferi RNA polymerase purified by affinity chromatography. The flow-through fraction of cell lysate (lane 1), first wash fraction (lane 2), last wash fraction (lane 3), and five elution fractions (lane 4-8) were diluted in 2x sample loading buffer, boiled, and separated by SDS-PAGE in a gradient polyacrylamide gel at 200 V for 30 min. Please click here to view a larger version of this figure.

Figure 2: Analysis of RpoD purification by gel electrophoresis. (A) Pooled flow-through fractions (lane 1 and lane 2), wash fractions (lanes 3-5), and elution fractions (lanes 6-10) from the purification of MBP-tagged RpoD by amylose resin affinity chromatography. (B) Protein mixture following the cleavage of MBP and RpoD using Factor Xa protease. (C) Flowthrough solution (lane 1) and elution fractions with an increasing gradient of NaCl (lanes 2-9) from heparin affinity chromatography to separate MBP and RpoD. In each gel presented, the samples were mixed with 2x loading buffer, boiled, and separated in a gradient polyacrylamide gel using SDS-PAGE at 200 V for 30 min. Please click here to view a larger version of this figure.

Figure 3: PCR generation of in vitro transcription templates. 500 bp sized amplicon containing flgB promoter (lane 1 and lane 2) and control PCR reactions containing amplicons of other promoter sites (lanes 3-4) are shown. Please click here to view a larger version of this figure.

Figure 4: Experimental arrangement and pipetting sequence plan. Please click here to view a larger version of this figure.

Figure 5: RpoD-dependent variability in phosphor image signal from RNA generated by in vitro transcription using B. burgdorferi RNA polymerase. In vitro transcription reactions contained 50 nM of RNA polymerase and varying levels of RpoD indicated above the bands²⁴. RNA was separated by a 10% TBE-urea gel, and a phosphor screen was used to capture the signal from radioactive decay over 16 h. This figure has been modified from Boyle et al.²⁴. Please click here to view a larger version of this figure.

Figure 6: RpoD-dependent variability in phosphor image signal from RNA generated by in vitro transcription. In vitro transcription contained 25 nM of RNA polymerase and varying levels of RpoD indicated above the bands. RNA was separated by a 15% TBE-urea gel, and a phosphor screen was used to capture the signal from radioactive decay over 16 h. The lower RNA polymerase concentration and higher background signal increased the difficulty of accurate measurement. Please click here to view a larger version of this figure.

Figure 7: Increasing radiolabeled RNA accumulation in in vitro transcription reactions with increasing RpoD levels. The signals detected in the phosphor screen imaging were analyzed by densitometry with no background subtraction. Please click here to view a larger version of this figure.

Table 1: Media recipes. Please click here to download this Table.

Table 2: FPLC settings. Please click here to download this Table.

Table 3: PCR reactions. Please click here to download this Table.

Table 4: 50 nM RNA polymerase experimental plan and volume calculation. Please click here to download this Table.

Table 5: 25 nM RNA polymerase experimental plan and volume calculation. Please click here to download this Table.

Table 6: Strains and oligonucleotides used in this protocol. Please click here to download this Table.

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 flexible and modifiable. The reactions are modular and built from frozen stocks and require little lead time once the experimental parameters have been chosen. Given sufficient stock, multiple experiments may be carried out in 1 day.

Obtaining active RNA polymerase is the most critical aspect of in vitro transcription reaction preparation. We recommend harvesting RNA polymerase from cells grown to the mid-logarithmic growth phase as described in this protocol. In purifying the RNA polymerase from any bacteria, the rate of growth has been noted as an important factor in purifying active RNA polymerase. RNA polymerases can accumulate post-translational modifications and may be associated with inhibitory transcription factors at the stationary phase of growth³¹. We have not succeeded in purifying high amounts of active RNA polymerase from B. burgdorferi cultures grown to stationary phase. The affinity chromatography approach, as compared to a physical separation approach³², is also beneficial for purification from B. burgdorferi because the organism reaches low density in culture, and accumulating large amounts of cell culture is relatively material intensive.

During purifications of other bacterial RNA polymerases, sigma factors can co-purify in significant quantities depending on the purification method and conditions³³^,³⁴^,³⁵^,³⁶. Co-purification of sigma factors can limit downstream experiments using alternative sigma factors, modified sigma factors, or a lack of sigma factors. Moreover, supplementation with the housekeeping sigma factor RpoD is beneficial for detecting activity regardless of initial co-purification of the sigma factor³⁷. Due to the flexibility of separating the sigma factor from RNA polymerase core, we consider it advantageous that sigma factors do not co-purify under the His-affinity chromatography purification conditions outlined here. It is possible that the purification conditions could be changed to yield more intact and active holoenzymes containing RpoD. For example, salt concentrations are likely to affect the stability of multiple subunit enzymes, and the effect of imidazole concentration on the stability of the B. burgdorferi RNA polymerase complex or its divalent cation incorporation is also unknown.

B. burgdorferi RNA polymerase enzymatic activity is sensitive to the type and quantity of divalent cations present²⁴. We found that manganese is required for maximal RNA polymerase activity, and magnesium appeared to contribute to activity to a lesser extent. Contaminating ions in water add a high amount of variability to RNA polymerase enzymatic activity. Consequently, highly pure water such as molecular biology grade, HPLC grade, and metal analysis grade H₂O with few metal ions present is essential for higher RNA polymerase activity in the protocol outlined here. RNA polymerases are also sensitive to pH and require a reducing environment during purification²⁷. Altered pH or the use of BME as a reducing agent has resulted in inactive RNA polymerase. We recommend using commercially available E. coli RNA polymerase as a control to ensure the proper construction of in vitro transcription assays²⁴. E. coli RNA polymerase will allow for testing of the viability of the nucleotides, the template construction method, radiolabel incorporation, RNA separation, and the signal detection method. However, it should be noted that B. burgdorferi RNA polymerase has specific divalent cation, buffer, and promoter requirements compared to E. coli.

There are several detection methods available for in vitro transcription reactions. Dye incorporation and molecular beacon assays are common alternatives to the radiolabel detection method³⁸^,³⁹. Radiolabeled nucleotide incorporation is currently the most sensitive RNA detection method. Alternative assays require more RNA polymerase to reach measurable signal levels compared to radiolabel assays. Additionally, dye incorporation and molecular beacon methods typically have more ways for signals to be erroneous. For example, DNase contamination can cause molecular beacons to release a signal in the absence of their target. Using radiolabel incorporation, the relative activity of the B. burgdorferi RNA polymerase enzyme can be detected even at low levels, which suits initial experimentation with RNA polymerase enzymes.

While the concentration of B. burgdorferi RNA polymerase enzyme included in the reaction is standardized by spectrophotometry, this measurement can be a source of variability. The specific activity of RNA polymerases can depend on the number of fully-formed active holoenzymes, which are a portion of the total protein mixture⁴⁰. For example, the molar quantity of 50 nM of RNA polymerase reported in our results was determined by spectrophotometry, but an analysis of the subunit quantities by quantitative western blotting revealed that the RpoA subunit was a limiting component in the number of potential RNA polymerase holoenzymes, and a maximum of only 21 nM of fully formed RNA polymerase was possible in our mixture²⁴. Experiments should always be conducted to control for batch-to-batch variation in RNA polymerase and other components of the in vitro transcription reaction.

In our representative experiment shown in Figure 5, the RpoD levels were altered, and a linear relationship between RpoD and the detected signal was demonstrated. In vitro transcription is a versatile method that can be easily altered using the experimental template presented (Table 4). For example, alternative sigma factors or alternative DNA templates can be easily tested²⁴. Alternative templates could include different promoter regions generated by PCR or plasmid purified from cells to introduce supercoiled and methylated DNA features. For a given level of RpoD, RNA polymerase, and template, differing levels of a transcription factor may be added to see inhibition or enhancement of activity from a promoter²³. The order of component addition and molar ratios within the in vitro transcription reaction can be altered such that transcription initiation complexes are formed prior to reaction initiation; these reactions could measure transcription elongation instead of transcription initiation⁴⁰. The in vitro transcription assay may be altered to suit a variety of investigations into B. burgdorferi biology.

Offenlegungen

The authors have nothing to disclose.

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

0.45 micron syringe filter	Thermo Scientific	726-2545	Step 1.7 and 2.3
50 mL conical tubes	MidSci	C50B	Step 1.3, and subsequent steps
50 mL high-speed centrifuge tubes	Thermo Scientific	3119-0050PK	Step 1.2
500 mL Centrifuge bottles	Thermo Scientific	3120-9500PK	Step 1.1
B-PER and instruction manual	Thermo Scientific	78248	Step 1.4 and 2.2
Calcium chloride	Fisher Scientific	10035-04-8	Step 2.6
Centrifugal filters 10 Kd cutoff	Millipore Sigma	UFC8010	Step 1.11 and 2.11
Cobalt resin and instruction manual	Thermo Scientific	89969	Step 1.9
Dithiothreitol	Acros Organics	426380500	Step 1.4 and subsequent steps
Dnase (Nuclease)	Millipore Sigma	70746	Step 1.4 and 2.2
Factor Xa Protease	Haematologic Technologies	HCXA-0060	Step 2.6
GE Typhoon 5 Phosphoimager	GE lifesciences	Multiple	Step 4.15
Gel Imager	Bio-Rad	Mutiple	Step 1.13 and subsequent protien quality check steps
H₂O for in vitro transcription	Fisher Scientific	7732-18-5	Step 3.2 and 3.3
high fidelity PCR kit	New England Biolabs	M0530S	Step 3.1
High-speed centrifuge	Eppendorf		Step 1.1, and subsequent steps
HiTrap Heparin HP 5 x 1 mL	Cytiva Life Sciences	17040601	Step 2.8
Imidazole	Sigma-Aldrich	56750-100G	Step 1.9
Lysozyme	Thermo Scientific	90082	Step 1.4 and 2.2
Magnesium chloride	Fisher Scientific	S25401	Step 4.1
Manganese chloride	Fisher Scientific	S25418	Step 4.1
Mini protean tetra cell	Bio-Rad	Mutiple	Step 1.13 and subsequent protien quality check steps
NP-40	Thermo Scientific	85124	Step 4.1
NTP mixture	Thermo Scientific	R0481	Step 4.1
PCR purification kit	Qiagen	28506	Step 3.2
PCR tubes	MidSci	PR-PCR28ACF	Step 1.12
PD-10 Sephadex buffer exchange column and instruction manual	Cytiva	17085101	Step 1.10 and 2.10 (gel filtration column)
pMAL Protein Fusion and Purification System Instruction manual	New England Biolabs	E8200S	Step 2.1
Polyacrylamide gels AnyKD	Bio-Rad	456-8125	Step 1.13 and subsequent protien quality check steps
Potassium glutamate	Sigma-Aldrich	G1251	Step 4.1
Protease inhibitor	Thermo Scientific	78425	Step 1.4 and 2.2
Radiolabeled ATP	Perkin Elmer	BLU503H	Step 4.2
RNA Loading Dye, (2x)	New England Biolabs	B0363S	Step 4.13
Rnase inhibitor	Thermo Scientific	EO0381	Step 4.1
Spectrophotometer	Biotek	Mutiple	Step 1.13 and subsequent protien quality check steps
TBE-Urea gels 10 percent	Bio-Rad	4566033	Step 4.14

Referenzen

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Boyle, W. K., Sorensen, H. N., Bourret, T. J. Essential Components of Borreliella (Borrelia) burgdorferi In Vitro Transcription Assays. J. Vis. Exp. (185), e64134, doi:10.3791/64134 (2022).