In Vitro Transcription and Immobilized Template Analysis of Preinitiation Complexes

Justin J. Lin1, Michael Carey1

1 University of California, Los Angeles, Los Angeles, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 12.14
DOI:  10.1002/0471142727.mb1214s97
Online Posting Date:  January, 2012
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Abstract

In the study of gene regulation, it is often necessary to employ functional assays that investigate the action or mechanism of specific promoters or enhancer‐binding factors and their role in transcription by RNA polymerase II. Although many assays measure the transcription of a gene under the control of an endogenous or model activator in vivo, it is often useful to recreate transcription in vitro in order to study specific regulatory mechanisms. In this unit, protocols are presented that will allow the investigator to perform in vitro transcription using preinitiation complexes assembled from cellular extracts on either naked DNA or chromatin templates. Curr. Protoc. Mol. Biol. 97:12.14.1‐12.14.19. © 2012 by John Wiley & Sons, Inc.

Keywords: in vitro transcription; chromatin reconstitution; immobilized template

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: In Vitro Transcription from Nuclear Extracts
  • Basic Protocol 2: PIC Capture Assay on Immobilized DNA Templates
  • Support Protocol 1: Primer Extension of In Vitro–Transcribed RNA
  • Support Protocol 2: Rapid Chromatin Assembly by Salt Dilution
  • Support Protocol 3: Preparation of HeLa Nuclear Extracts
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: In Vitro Transcription from Nuclear Extracts

  Materials
  • DNA template (e.g., 602‐bp linear chromatin template containing the G5E4T promoter at a starting concentration of 5 ng/µl)
  • 200 ng/µl activator protein in Buffer D
  • 10 mg/ml bovine serum albumin (BSA) in ddH 2O
  • Buffer D (0.1 M KCl; see recipe)
  • 100 µg/ml nonspecific competitor DNA (e.g., pGEM3 plasmid vector used to clone G5E4T template promoter)
  • 0.1 M MgCl 2
  • 1 mM acetyl‐coenzyme A (acetyl‐CoA)
  • HeLa nuclear extract (see protocol 5)
  • 25 mM NTPs (25 mM each, ATP, UTP, CTP, and GTP)
  • Transcription stop buffer (see recipe)
  • 10 mg/ml proteinase K
  • 50 mg/ml carrier yeast tRNA or nucleic acid co‐precipitant (e.g., Novagen Pellet Paint Co‐precipitant, EMD/Millipore)
  • Phenol
  • Chloroform
  • Ethanol (100%)
  • Dry Ice
  • Drawn‐out glass Pasteur pipet (>0.5 mm diameter)
CAUTION: Take appropriate care when using phenol and chloroform, and consult the materials safety data sheets (MSDSs) before use.

Basic Protocol 2: PIC Capture Assay on Immobilized DNA Templates

  Materials
  • Streptavidin M‐280 Dynabeads (Invitrogen 112‐05D)
  • 2× BW Buffer (see recipe)
  • DNA template (e.g., 602‐bp linear chromatin template containing the G5E4T promoter at a starting concentration of 5 ng/µl)
  • Buffer D (0.1 M KCl; see recipe)
  • 100 µg/ml nonspecific competitor DNA (e.g., pGEM3 plasmid vector used to clone G5E4T template promoter)
  • 1% Nonidet P‐40 (NP‐40) or equivalent
  • 0.1 M MgCl 2
  • 10 mg/ml bovine serum albumin (BSA) in ddH 2O
  • 200 ng/µl activator protein in Buffer D
  • 1 mM acetyl‐coenzyme A (acetyl‐CoA)
  • HeLa nuclear extract (see protocol 5)
  • Magnetic Particle Concentrator (MPC; Invitrogen 120.26, 120.27, 120.28D)
  • End‐over‐end rotator
  • Additional reagents and equipment for SDS‐PAGE (unit 10.2) and immunoblotting (unit 10.8)

Support Protocol 1: Primer Extension of In Vitro–Transcribed RNA

  Materials
  • 10 µM reverse transcription primer
  • 10 U/µl T4 polynucleotide kinase (PNK) with 10× reaction buffer (NEB M0201)
  • [32P]γ‐ATP (>5000 Ci/mmol; Perkin Elmer BLU002Z500UC)
  • 100 mM DTT
  • In vitro−transcribed mRNA (see protocol 1)
  • 2× hybridization buffer (see recipe)
  • 1 M ammonium acetate, pH 7 (neutral salt)
  • Isopropyl alcohol
  • Nucleic acid co‐precipitant (e.g., Novagen Pellet Paint; optional)
  • 75% (v/v) ethanol, ice cold
  • 5× reverse transcriptase (RT) buffer (Promega M515A1)
  • 10 mM dNTP mix (10 mM each dATP, dCTP, dGTP, dTTP)
  • RNAsin (Promega N2511)
  • Avian myeloblastosis virus (AMV) reverse transcriptase (Promega M5108)
  • 2× formamide loading dye (see recipe)
  • G50 desalting column (GE Biosciences)
  • Drawn‐out Pasteur pipet
  • Geiger counter (optional)
  • Additional reagents and equipment for urea/PAGE (unit 2.5) and imaging ( appendix 3A)

Support Protocol 2: Rapid Chromatin Assembly by Salt Dilution

  Materials
  • 2 and 5 M NaCl
  • 1 mg/ml bovine serum albumin (BSA)
  • DNA template (in H 2O or standard Tris⋅EDTA, pH 7.5‐8.0)
  • Histone octamer (e.g., Workman et al., ; Luger et al., )
  • Assembly buffer (see recipe)
  • Additional reagents and equipment for PAGE electrophoretic mobility shift assay (EMSA; unit 10.2)

Support Protocol 3: Preparation of HeLa Nuclear Extracts

  Materials
  • HeLa cells in culture
  • 1× PBS
  • Buffer A (see recipe)
  • Buffer C (see recipe)
  • Buffer D (0.1 M KCl; see recipe)
  • Dry ice
  • Refrigerated centrifuge (e.g., Sorvall) with graduated centrifuge tubes or bottles
  • Dounce homogenizer with loose‐ and tight‐fitting pestles
  • Magnetic stirrer or end‐over‐end rotator
  • Microscope
  • Dialysis tubing, 12 kDa MWCO
  • Conductivity meter
  • Additional reagents and equipment for cell counting with Trypan blue staining ( appendix 3F), dialysis ( appendix 3C), and Bradford protein assay (unit 10.1)
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Figures

  •   FigureFigure 12.14.1 In vitro transcription from nuclear extracts. (A) Schematic of in vitro transcription assayed by primer extension. (B) Example of an in vitro transcription experiment determining the optimal levels of both nuclear extract and GAL4‐VP16 activator on the model promoter, G5E4T.
  •   FigureFigure 12.14.2 PIC capture on an immobilized template. (A) Schematic of PIC capture and purification from nuclear extract using the immobilized model promoter, G5E4T. (B) Immunoblots of a time‐course assay of stepwise PIC assembly demonstrates the kinetics of co‐activator and general transcription factor (GTF) recruitment from nuclear extract.
  •   FigureFigure 12.14.3 Chromatin assembly by rapid salt dilution. Schematic of chromatin assembly reaction (A), followed by a native PAGE electrophoretic mobility shift assay (EMSA) of chromatin assembly reactions with an octamer:DNA ratio titration (B). Two separate reactions for each titration point are shown. For comparison, free DNA inputs are run on the right side of the gel. DNA is visualized by SYBR‐green staining.

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Literature Cited

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