In Vitro Transcription Assays and Their Application in Drug Discovery

Summary

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

Abstract

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.

Introduction

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 expression<....

Protocol

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

1. Obtain the Proteins Required for Transcription assay.
   1. For the experiment described here, overproduce and purify

Results

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 resi.......

Discussion

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........

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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,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