In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.
In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.
Transcription is the process in which RNA is synthesized from a specific DNA template. In eukaryotic cells there are three distinct RNAPs: RNAP I transcribes rRNA precursors, RNAP II is responsible for the synthesis of mRNA and certain small nuclear RNAs, and the synthesis of 5S rRNA and tRNA is performed by RNAP III. In bacteria, there is only one RNAP responsible for the transcription of all classes of RNA. There are three stages of transcription: initiation, elongation and termination. Transcription is one of the most highly regulated processes in the cell. Each stage in the transcription cycle represents a checkpoint for the regulation of gene expression1. For initiation, RNAP has to associate with a sigma factor to form holoenzyme, which is required to direct the enzyme to specific sites called promoters2 to form an open promoter complex. Subsequently, a large suite of transcription factors are responsible for the regulation of RNAP activities during the elongation and termination phases. The transcription factor examined here is the highly conserved and essential protein, NusA. It is involved in regulating transcription pausing and termination, as well as anti-termination during rRNA synthesis3-5.
In vitro transcription assays have been developed as powerful tools to study the complex regulatory steps during transcription6. In general, a linear fragment of DNA including a promoter region is required as the template for transcription. The DNA template is usually generated by PCR or by linearizing a plasmid. Purified proteins and NTPs (including one radiolabeled NTP for detection purposes) are then added and product analyzed following the required period of incubation. Using appropriate templates and reaction conditions, all stages of transcription have been examined using this approach which has enabled detailed molecular characterization of transcription over the last half century7. In combination with information on the 3-dimensional structure of RNAP it has also been possible to probe the molecular mechanism of transcription inhibition by antibiotics and antibiotic leads, and use this information in the development of new, improved drugs8-10.
In this work we provide examples of how transcription assays can be used to determine the mechanism of regulation by transcription elongation/termination factor NusA, and how the mechanism of action of a novel transcription initiation inhibitor lead can be determined.
Caution: Experiments involve the use of the radioactive isotope 32P and no work should be undertaken until all appropriate safety conditions have been met. Generally personnel are required to attend a safety course and undergo supervised practice prior to experiments using radioactive reagents. Please wear personal protection equipment (thermoluminescent dosimeter, safety glasses, gloves, radiation room lab coats, full length pants, closed-toe shoes) when performing the reaction.
1. Preparation of Assay Materials
2. Preparation of RNA Sequencing Gel
3. Transcription Reaction
4. RNA Gel Running and Development
Transcription efficiency can be determined by measuring the level of radiation in the bands at different time points. The pausing assay to test the function of NusA factor enabled visualization of the pause, termination, and run-off products (Figure 1A). In the presence of the N-terminal domain of NusA (NusA NTD; amino acid residues 1-137), appearance of the RNA products was significantly delayed compared to the control experiment lacking NusA. Deletion of amino acid residues 104-137 (Helix 3) of the NusA fragment, or alteration to alanine of a series of amino acids (residues K36, K37, R104, Q108, K111, Q112, Q116, and R119) resulted in the complete loss of NusA pause activity. Helix 3 residues R104 and K111 were shown to be essential for NusA NTD pause activity and with the R104A and K111A constructs giving similar pause half-life values to the control where NusA NTD was absent (Figure 1B). It should be noted that deletion of Helix 3 or alteration of these two amino acid residues has no effect on NusA binding to RNAP15. The result suggested that R104 and K111 were the key amino acids required for regulation of NusA pause activity.
Similarly, as shown in Figure 2A, the dose-response curve demonstrated the inhibition of in vitro transcription (multi-round) using different concentrations of a newly identified bacterial transcription inhibitor C514. C5 was identified following an in silico screen from project to rationally target the interaction of σ70 with its major binding site on RNAP called the Clamp-Helix region18. With addition of C5 into the transcription reaction, this compound can compete against σ70 for binding to RNAP, and thus inhibited transcription. Mechanistically, addition of C5 to RNAP followed by σ70 factor to the reaction mixture was significantly more efficient for transcription inhibition than the experiment using C5 after the formation of RNAP holoenzyme (Figure 2B). These results demonstrated that σ70 couldn't displace C5 bound to RNAP, or that C5 could not efficiently displace σ70 bound to RNAP in a single-round assay.
Figure 1: Effects of Mutant NusA NTD in transcription. (A) Transcription pause assays without NusA, using NusA NTD (+ NusA), NusA NTD with helix 3 deleted (Δ Helix), and NusA NTD with alanine substitutions at residues K36, K37, R104, Q108, K111, Q112, Q116, and R119 (All Ala). P, pause site; T, termination site; RO, run-off. Wedges above the gel demonstrate the time points sampled (0.5, 1, 1.5, 2, 5 and 10 min)15; (B) Pause activity of half-lives of mutant NusA NTD relative to wild-type protein. Average of 3 experiments with SD. This figure has been modified from Nucleic Acids Res. 43 (5), 2829-2840 (2015). Please click here to view a larger version of this figure.
Figure 2: Transcription inhibition by compound C5. (A) Inhibition curve of E. coli RNAP transcription by compound C5 in a multi-round assay14. Percent inhibition of transcription was determined by the RNA product relative to a control (no C5 present) against the concentration of C5 used in the reaction. (B) Percent inhibitory activity of compound C5 in single-round transcription assays with addition of C5 before (complex + σ70; gray) and after (Holoenzyme + C5; black) holoenzyme formation. This figure has been modified from ACS Infect. Dis. 2, 39-46 (2016). Please click here to view a larger version of this figure.
In all organisms, transcription is a tightly regulated process. In vitro transcription assays have been developed to provide a platform for testing the effects of transcription factors, small molecules and transcription inhibitors. In this method paper, an assay for general bacterial transcription was described. Transcription assays combined with sequencing gel electrophoresis of transcripts are very important for mechanistic studies as they allow visualization of all the transcription products along a timeline. As a result, this method is capable of identifying the influence of minor changes in reaction conditions and revealing a plausible mechanism of action. Furthermore, it becomes feasible to design specific transcription assays with regard to the function of transcription factors with unknown mechanisms of action.
In this paper we also described the method to confirm the mechanism of action of an inhibitor of RNAP holoenzyme formation. The method described here can display the change of each RNA transcript with time, and with appropriate modification can enable the testing of antibacterial agents against each step of transcription as appropriate. The multi-round assay can be used as a relative simple screening method to identify new transcription targeting inhibitors, while the single round assay can be used to study the mechanism of inhibitors, such as competitive/non-competitive properties. Note that small molecular aggregation can cause false positive hits as non-competitive inhibitors in the drug discovery process and the single round mechanistic study may provide a comprehensive approach for hit-to-lead evaluation. As a result, this controlled transcription process is capable of precisely revealing how an inhibitor affects transcription, which can help with rationalized de novo drug design. Together with high-resolution information on inhibitor binding sites by X-ray crystallographic studies, structure-based rational drug design will be highly amenable for application in drug discovery targeting transcription.
Although high-throughput results cannot be obtained with in vitro transcription assays, they are extremely useful in dissecting the precise mechanisms associated with the control of transcription. The components and assay conditions could be readily modified for individual needs. Various factors (e.g., NusA)4, 15, small molecules (e.g., ppGpp)19 and/or transcription inhibitors (e.g., rifampicin)20 of interest can be added to test their functions in transcription regulation. The DNA template should be individually designed, with careful choice of strong promoters, pause sequence and terminators. In order to achieve high reproducibility of the assay, proteins must be highly purified and free from residual RNase activity, and the transcription buffer, DNA template, and stock solution of NTP substrates must be made in DEPC treated water. The use of radioactive NTPs was described in this paper, however fluorescent nucleotides can also be employed21. Quality of the DNA template is critical for the success of transcription assay. Sufficient spacing between the promoter/pause/terminator must be included, allowing the various transcription products to be properly separated and examined. The sequencing gel must be properly poured and relatively fresh TBE buffer must be used for electrophoresis to achieve highest possible resolution of RNA transcripts. If degradation of transcription product is noticed, high quality RNase inhibitor must be incorporated in the transcription reaction mixture. Tubes and pipette tips must be double autoclaved and gloves must be worn at all times when performing the assay, in order to minimize introduction of RNase.
The authors have nothing to disclose.
This work acknowledges a Faculty Early Career Grant from the University of Newcastle (CM).
Obtain the proteins required for transcription assay | |||
E. coli RNAP | Epicentre | S90250 | |
Preparation of DEPC-treated water | |||
diethyl pyrocarbonate (DEPC) | Sigma-Aldrich | D5758 | |
RNase-free water | |||
Ambion Nuclease-Free Water | ThermoFisher | AM9937 | |
DNA template preparation | |||
Wizard Plus SV Minipreps DNA Purification System | Promega | A1330 | |
ACCUZYME Mix | Bioline | BIO-25028 | |
PCR primers | |||
Wizard SV Gel and PCR Clean-Up System | Promega | A9281 | |
NanoDrop 3300 fluorospectrometer | Thermo Scientific | ND-3300 | |
NTP Preparation | |||
ATP | Sigma-Aldrich | A6559 | |
UTP | Sigma-Aldrich | U1006 | |
GTP | Sigma-Aldrich | G3776 | |
CTP | Sigma-Aldrich | C9274 | |
High Purity rNTPs | GE Healthcare | 27-2025-01 | |
α-32P UTP | PerkinElmer | BLU007C001MC | Radioactive compound |
RNA ladder preparation | |||
Novagen Perfect RNA Marker Template Mix 0.1–1 kb | Millipore | 69003 | |
HEPES | Sigma-Aldrich | H7006 | |
Sodium chloride | Sigma-Aldrich | S7653 | |
Magnesium chloride | Sigma-Aldrich | M8266 | |
DTT | Sigma-Aldrich | DTT-RO | |
T7 RNAP | Promega | P2075 | |
Gel preparation | |||
Sequi-Gen GT nucleic acid sequencing cell | Bio-Rad | 165-3804 | |
Sigmacote | Sigma-Aldrich | SL2 | |
urea | Sigma-Aldrich | U6504 | |
tris(hydroxymethyl)aminomethane | Sigma-Aldrich | 154563 | |
boric acid | Sigma-Aldrich | B7901 | |
ethylenediaminetetraacetic acid | Sigma-Aldrich | ED | |
40% Acrylamide/bis-acrylamide | Sigma-Aldrich | A9926 | |
ammonium persulfate | Sigma-Aldrich | A3678 | |
N,N,Nʹ′,Nʹ′-Tetramethylethylenediamine (TEMED) | Sigma-Aldrich | T9281 | |
N,N,N”,N”-Tetramethylethylenediamine (TEMED) | Sigma-Aldrich | T9281 | |
Transcription Assay | |||
Potassium chloride | Sigma-Aldrich | P9541 | |
glycerol | Sigma-Aldrich | G5516 | |
rifampicin | Sigma-Aldrich | R3501 | |
formamide | Sigma-Aldrich | F9037 | |
bromophenol blue | Sigma-Aldrich | B0126 | |
xylene cyanol | Sigma-Aldrich | X4126 | |
heparin | Sigma-Aldrich | 84020 | |
RNasin Ribonuclease Inhibitor | Promega | N2511 | |
Transcription buffer | |||
Tris base | Sigma-Aldrich | T1503 | |
Potassium chloride | Sigma-Aldrich | P9541 | |
Magnesium chloride | Sigma-Aldrich | M2393 | |
DTT | Sigma-Aldrich | DTT-RO | |
glycerol | Sigma-Aldrich | G5516 | |
Filter paper | |||
Whatman 3MM Chr Chromatography Paper | Fisher Scientific | 05-714-5 | |
Radioactive decontaminant | |||
Decon 90 | decon | decon90 | |
Gel Treatment | |||
Typhoon Trio+ imager | GE Healthcare Life Sciences | 63-0055-89 |