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.

Run off transcription assay

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 |

Vo et al.

Steady state transcription assay

20-50μL reaction mixtures (prepped on ice)

• 30 nM promoter template
• 150 mM KCl
• 100μM each of four NTPs
• [γ-<sup32P]ATP (~5cpm/fmol) in buffer
  • 40 mM Tris-HCl pH8
  • 10 mM MgCl₂
  • 10 mM β-mercaptoethanol
  • 10 μg/mL acetylated BSA

1. Add E. coli RNA polymerase to final concentration of 30 nM
2. Incubate at 37°C
3. Remove 5-10 μL samples at 0-60 mins
4. Mix with equal volume of formamide loading buffer
   • 80% (v/v) freshly deionized formamide
   • 1X TBE
     • 89 mM Trisma base
     • 89 mM boric acid
     • 2.5 mM Na₂EDTA (pH 8.3)
   • 10 mM Na₂EDTA
   • 0.025% (w/v) xylene cyanol
5. Heat samples to 100°C for 3 mins
6. Load onto prerun gel
7. Fractionate by gel electrophoresis on 23% (38:2) polyacrylamide-7M urea gels in salt gradient buffer.
8. Continue electrophoresis at constant power of 1.5 W/cm until XC has migrated 17 cm from wells
9. Expose and scan in phosphorimager

Reference

4. Vo NV, Hsu LM, Kane CM, and Chamberlin MJ. In vitro studies of transcript initiation by Escherichia coli RNA polymerase. 3. Influences of individual DNA elements within the promoter recognition region on abortive initiation and promoter escape. Biochemistry. 2003 Apr 8;42(13):3798-811. DOI:10.1021/bi026962v |