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.
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.
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.
1. Purification of the RNA polymerase and preparation of RNA polymerase stock
2. Purification of recombinant RpoD and preparation of RpoD stock
3. Preparation of DNA template stock
4. Perform in vitro transcription with the incorporation of radiolabeled nucleotides
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 OD600nm 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.
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.
The authors have nothing to disclose.
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.
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 |