Knight:In vitro transcription

Purpose

In vitro transcription using Escherichia coli RNA polymerase.

Materials

• E. coli RNA Polymerase Sigma-Saturated Holoenzyme from Epicentre - (protocol)

Reaction conditions

Epicentre

Functional test

50 μL reaction

• 1X E. coli RNA polymerase transcription buffer
  • 0.04 M Tris-HCl (pH 7.5)
  • 0.15 M KCl
  • 10 mM MgCl₂
  • 0.01% Triton® X-100
• 2mM DTT
• 0.25mM NTPs
• 1μg T7 D111 DNA as template

Assay test

50 μL reaction

• 1X E. coli RNA polymerase transcription buffer
  • 0.04 M Tris-HCl (pH 7.5)
  • 0.15 M KCl
  • 10 mM MgCl₂
  • 0.01% Triton® X-100
• 10 mM DTT
• 0.5 mM NTPs
• 1μg T7 D111 DNA as template

Szalewska-Palasz et al.

25 μL reaction

• Buffer M
  • 20 mM Hepes, pH 8.0
  • 5 mM magnesium acetate
  • 4 mM DTT
  • 1 mM EDTA
  • 1mM ATP
  • BSA (5mg/mL)
  • 0.2% Triton X-100
  • 5% glycerol
• 5 μg template DNA

Procedure

1. Add repressor
2. Incubate 5min at 37°C
3. Transfer samples to ice bath
4. Add 1 unit E. coli RNAP from Epicentre
5. Add 150 μM CTP and GTP
6. Add 1 mM ATP
7. Add 15 μM UTP
8. Add [α-³²P]UTP to 1mCi/mL
9. Incubate samples at 37°C for 12.5 mins
10. Stop reaction with equal volume of
    • BSA (1.2mg/mL)
    • 0.1 mM EDTA, pH 8.0
    • 5.1 M ammonium acetate
11. Transfer to ice bath
12. Precipitate RNA with 2 volumes 96% ethanol in liquid nitrogen
13. Centrifuge in microcentrifuge at maximum speed for 30 mins
14. Dry pellet
15. Resuspend in 20 μL of
    • 98% formamide
    • 0.25% bromophenol blue
    • 0.25% xylene cyanol
16. Incubate at 65°C for 5 mins
17. Electrophorese in 6% polyacrylamide gel containing 46% urea in TBE buffer at 30mA
18. Dry the gel
19. Visualize RNA bands by autoradiography and quantify via densitometry

Reference

1. Szalewska-Pałasz A, Wegrzyn A, Błaszczak A, Taylor K, and Wegrzyn G. DnaA-stimulated transcriptional activation of orilambda: Escherichia coli RNA polymerase beta subunit as a transcriptional activator contact site. Proc Natl Acad Sci U S A. 1998 Apr 14;95(8):4241-6. DOI:10.1073/pnas.95.8.4241 |

Galan et al

(Run off transcription assay)

9 μL reaction

• 5nM DNA (supercoiled plasmid)
• 100nM CRP
• 100nM HpaR or buffer B

• Buffer B
  • 40 mM Tris-HCl pH 8.0
  • 10 mM MgCl₂
  • 100 mM KCl
  • 200 μM cAMP
  • 500 μg/mL acetylated BSA

Procedure

1. Incubate reaction at room temperature for 20 mins
2. Add 3 μL RNAp at 375 nM in buffer B
3. Incubate 5 mins at 37°C
4. Start elongation with 3 μL of prewarmed mixture in buffer B of
   • 1mM ATP
   • 1mM GTP
   • 1mM CTP
   • 50μM UTP
   • 1μCi [α-³²]UTP
   • 500 μg/mL heparin
5. Incubate 5 mins at 37°C
6. Add 12μL loading buffer containing 1% SDS
7. Heat to 70°C
8. Electrophorese samples on 7% sequencing gels
9. Quantify using phosphorimager

Reference

2. Galán B, Kolb A, Sanz JM, García JL, and Prieto MA. Molecular determinants of the hpa regulatory system of Escherichia coli: the HpaR repressor. Nucleic Acids Res. 2003 Nov 15;31(22):6598-609. DOI:10.1093/nar/gkg851 |

Marschall et al.

1. Used either supercoiled or linear template
2. 5 μL of 20nM DNA template in potassium glutamate solution
   • 40 mM Hepes (pH 8.0)
   • 10 mM magnesium chloride
   • 100 mM potassium glutamate
   • 200 μM cAMP
   • 500 μg/mL acetylated bovine serum albumin
3. Add 5 μL of 400 nM Lrp or buffer
4. Add 5 μL of 400 nM Crp or buffer
5. Incubate at room temperature for 15 mins
6. 7μL of mixture was incubated at 37°C for 5 mins
7. Add 3.5 μL RNAp at 260 nM
8. Incubate for 5 mins at 37°C
9. Start polymerization by adding 3.5 μL prewarmed mixture containing
   • 1mM ATP
   • 1mM GTP
   • 1mM CTP
   • 50μM UTP
   • 500 μg/mL heparin
   • 1μCi [α-³²]UTP
10. Incubate 5 mins (what temp?)
11. Stop reaction by adding 20mM EDTA in formamide containing xylene cyanol and bromophenol blue
12. Heat to 65°C
13. Electrophorese samples on 7% sequencing gels
14. Autoradiograph and quantify using phosphorimager

Reference

3. Marschall C, Labrousse V, Kreimer M, Weichart D, Kolb A, and Hengge-Aronis R. Molecular analysis of the regulation of csiD, a carbon starvation-inducible gene in Escherichia coli that is exclusively dependent on sigma s and requires activation by cAMP-CRP. J Mol Biol. 1998 Feb 20;276(2):339-53. DOI:10.1006/jmbi.1997.1533 |