The in vitro transcription assay system for Borreliella burgdorferi provides a biochemical tool for investigators to study gene regulatory mechanisms and factors that govern the enzymatic activity of RNA polymerase. The system allows us to test how transcription factors, co-factors, salt concentrations, and pH impact Borreliella burgdorferi RNA polymerase function, which contributes to our overall understanding of gene regulatory mechanisms. This powerful technique can also allow us to screen for drugs that selectively inhibit RNA polymerase, opening the door to developing novel drugs to treat Lyme borreliosis.
To begin, collect the cell pellet from two to four liters of Borreliella burgdorferi RpoC-His10X cultured in BSKII medium in a microaerophilic environment to a density of two to four times 10 constant per milliliters. Pellet the cells at 10, 000 times G for 30 minutes at four degrees Celsius in 500 milliliters centrifugal bottles and discard the supernatant. Then, resuspend the cells in 30 milliliters of ice-cold HN buffer, repeat the centrifugation step, and decant the supernatant.
While working, maintain the cells, lysate, and purified proteins at four degrees Celsius or on ice unless freezing. Keep RNA polymerase in a reducing environment by adding freshly prepared dithiothreitol at a concentration of two-millimolar to every buffer used and maintain pH 8.0. Prepare lysate from the Borreliella burgdorferi cell pellet using a commercial bacterial lysis kit.
Resuspend the pellet in 10 to 15 milliliters of B-PER solution without protease inhibitor and allow the lysis to proceed for five minutes on ice. Add a protease inhibitor cocktail to the lysis solution and proceed with three rounds of sonication. Now clarify the cell lysate by centrifugation and filtration using a 50-milliliter centrifuge tube.
Add cobalt column loading buffer to the solution, making the total volume up to 30 milliliters. Pellet the cell debris by centrifugation at 20, 000 times G for 30 minutes. Later, filter the supernatant using a 0.45-micrometer syringe filter and dilute the lysate and cobalt column loading buffer to a final dilution ratio of 1:10.
Perform affinity chromatography on the clarified cell lysate supernatant using a cobalt or nickel resin column following the manufacturer's instructions. Save the flow-through, wash, and eluted samples for analysis. Immediately exchange the RNA polymerase solution buffer solution with the storage buffer solution using a buffer exchange column following the manufacturer's instructions.
Then, concentrate the RNA polymerase to a concentration of 0.2 to 0.4 milligram per milliliter using 10-kilodaltons-cutoff centrifugal filter units. Determine the concentration by spectrophotometer and prepare the freezing stocks. Aliquot 20 to 50 microliters volumes of RNA polymerase freezing stocks in PCR tubes and store the RNA polymerase at minus 80 degrees Celsius.
Prepare the work surfaces, including the radiation bench, to reduce radiation contamination. Thaw fresh RNA polymerase and RpoD stocks on ice and mix all the thawed frozen stocks thoroughly prior to pipetting Prepare a master reaction mixture in a separate NTP mixture for radiolabeled nucleotides according to the experimental requirements. Dispense the control reactions in experimental sets into PCR tubes.
After dispensing water, reaction mix, RNA polymerase, and RpoD into designated PCR tubes, mix the reagents by pipetting. Transfer the prepared materials to a radiation bench, add alpha-32P-labeled ATP to NTP, and mix by gentle pipetting. Add NTP to the tubes containing the in vitro transcription reaction mixtures and start the in vitro transcription reaction by adding a DNA template.
Mix the reaction volume by gently pipetting and incubate the tubes at 37 degrees Celsius for five minutes in a thermocycler or heat block. Remove the reactions from the thermocycler or heat block and stop the in vitro transcription reactions by adding an equal volume of 2X RNA loading dye containing 50%formamide to the reaction mixture. Denature of the enzymes by incubating reactions at 65 degrees Celsius for five minutes in a thermocycler or heat block.
Using gel electrophoresis, separate the in vitro transcribed RNA in 10%to 15%TBE urea polyacrylamide gels at 180 volts for 30 to 45 minutes. After removing any portion of the gel containing unincorporated radiolabeled ATP, expose the gel to the phospho screen overnight and image the radiolabeled RNA using a phosphoscreen imager. The SDS-PAGE showed three bands corresponding to three peptides, RpoC, RpoB, and RpoA of RNA polymerase complex.
A peptide of 115 kilodaltons matching the size of the MBP-tagged recombinant Borreliella burgdorferi RpoD was also observed. Cleavage of the protein mixture with factor XA protease led to the generation of two significant products, RpoD and MBP. The promoter site of Borreliella burgdorferi FLGB was generated by PCR.
A twofold dilution of RpoD concentration gives rise to a lower level of the accumulated RNA product. A low RNA polymerase concentration gives fewer RNA products across the range of RpoD concentrations. The densitometry signal indicated a linear relationship between the amount of RpoD present in the reaction in both experiments.
RNA polymerase enzymatic activity is sensitive to a variety of factors during the purification, storage, and experimental process. Fresh enzyme and reagents, along with good planning, give the best possible chance of success in this protocol.
