Run-off transcription

Run-off transcription is an assay in molecular biology which is conducted in vitro to identify the position of the transcription start site (1 base pair upstream) of a specific promoter along with its accuracy and rate of in vitro transcription. ^{[1] [2] [3]} Run-off transcription can be used to quantitatively measure the effect of changing promoter regions on in vitro transcription levels, ^{[1] [2] [4]} Because of its in vitro nature, however, this assay cannot accurately predict cell-specific gene transcription rates, unlike in vivo assays such as nuclear run-on. ^{[1] [2]}

History and Background

Paul Berg in 1980

Michael Chamberlin (biologist), who passed away in his sleep in November, 2025, was the first person to isolate RNA polymerase from Escherichia coli as a graduate student at Stanford University with the late Paul Berg and revolutionized the understanding of transcription process. ^[5]

Principle

To perform a run-off transcription assay, a gene of interest, including the promoter, is cloned into a plasmid. ^[4] The plasmid is digested at a known restriction enzyme cut site downstream from the transcription start site such that the expected mRNA run-off product would be easily separated by gel electrophoresis. ^{[1] [2] [4]} DNA needs to be highly purified prior to running this assay. ^{[1] [2]} To initiate transcription, radiolabeled UTP, the other nucleotides, and RNA polymerase are added to the linearized DNA. ^{[1] [2]} Transcription continues until the RNA polymerase reaches the end of the DNA where it simply “runs off” the DNA template, resulting in an mRNA fragment of a defined length. ^{[1] [2]} This fragment can then be separated by gel electrophoresis, alongside size standards, and autoradiographed. ^{[1] [2] [4]} The corresponding size of the band will represent the size of the mRNA from the restriction enzyme cut site to the transcription start site (+1). ^[4] The intensity of the band will indicate the amount of mRNA produced. ^[4] Additionally, it can be used to detect whether or not transcription is carried out under certain conditions (i.e. in the presence of different chemicals). ^[6]

Procedure

Preparing DNA template: DNA templates for transcription assay are either plasmids or dsDNA fragments constructed using synthesized oligonucleotides. The circular DNA template that contains the promoter sequence (T7 promoter) is linearized using Restriction endonuclease that cuts downstream of the region to be transcribed. It ensures the RNA polymerase will transcribe the DNA until it runs off the end of the linearized DNA molecule.

Assembly of the transcription assay: The linearized DNA molecule is mixed with Tris-HCl, MgCl₂, Spermidine, DTT and nucleotides (ATP, GTP,CTP, UTP). The reaction is incubated at 37°C for T7. Radiolabeled or fluorescently labeled nucleotides can be added too. For large scale assays, addition of Ribonuclease inhibitor and inorganic pyrophosphatases are recommended to improve the quality and yield of the transcripts. ^[7] After transcription, excess DNA template could be removed by DNase treatment.

Applications

• Run-off transcription microarray analysis (ROMA): Bacterial gene expression can be regulated in many levels including activating or repressing DNA-binding transcription factors at the transcription initiation site or the RNA polymerase containing different Sigma factors. Purified RNA polymerase holoenzyme is used on fragmented genomic DNA for in vitro transcription and mRNA transcripts are identified by Microarray hybridization analysis. Then ROMA allowed investigation of direct effects of different sigma factors like overlapping sigma 70 and sigma 38 without regulatory protein. ^[8]  ROMA is limited by lack of single nucleotide resolution and transcriptional read-through at convergently originated genes which can lead to false positive signals. ^[9]
• Run-off transcription/RNA-Seq (ROSE): To overcome the limitations of ROMA, scientists developed ROSE, a bottom-up approach aimed to assemble the transcriptional machinery to complement top-down in vivo transcriptome profiling in E. coli K-12 MG1655 genomic DNA. It is a genome-wide in vitro transcription with isolated RNA polymerase, ribonucleotides and genomic DNA. A library of native 5'-end specific transcript is prepared to provide distinct read of the transcription start point. It enables the detection of promoter sequences with single nucleotide resolution. ^[10]

Limitations

• Limited processivity ^[11]
• High salt sensitivity ^[11]
• Undesired products resulting from abortive synthesis ^[12]
• Addition of a non-base-paired nucleotide at the 3' end of the run-off transcript ^{[13] [14]}

Recent Advancements

Figure: Adsorption of a cyanophage onto a marine prochlorococcus.

To overcome the limitations of using T7 RNA polymerase, bacteriophage T3 and SP6 RNA Polymerase, scientists made extensive efforts to improve the 3' homogeneity of T7 transcripts including the modifications of DNA template, and the attachment of ribozymes to the 3' end of the desired RNAs. A marine organism named Cyanophage Syn5, a single subunit RNA polymerase was characterized for this purpose. Syn5 RNA polymerase can recognize a relatively short promoter sequence, a high tolerance to salt, high processivity because of two promoter sequences in its genome and most importantly, it has much higher homogeneity of the 3'-termini of its RNA products. It can produce precise run-off transcripts lacking non-based additional nucleotides which is crucial for in vitro synthesis of tRNAs, RNA probes, and RNA primers. Syn5 RNA polymerase exhibits greater stability than T7 RNA polymerase over 4 hours of incubation at 37°C. ^[15]

References

1. Loewenstein, P. M.; Song, C. Z.; Green, M (2007). "The Use of in Vitro Transcription to Probe Regulatory Functions of Viral Protein Domains". Adenovirus Methods and Protocols. Methods in Molecular Medicine. Vol. 131. pp. 15–31. doi:10.1007/978-1-59745-277-9_2. ISBN   978-1-58829-901-7. PMID   17656772.
2. "Run-off Transcription". Molecular Station. Archived from the original on April 22, 2014. Retrieved April 16, 2014.
3. Lelandais, C; Gutierres, S; Mathieu, C; Vedel, F; Remacle, C; Maréchal-Drouard, L; Brennicke, A; Binder, S; Chétrit, P (1996). "A promoter element active in run-off transcription controls the expression of two cistrons of nad and rps genes in Nicotiana sylvestris mitochondria". Nucleic Acids Research. 24 (23): 4798–804. doi:10.1093/nar/24.23.4798. PMC   146301 . PMID   8972868.
4. Allison, Lizabeth. "Fundamental molecular biology, chapter 11" (PDF). BlackWell Publishing. Retrieved April 18, 2014.
5. "In Memoriam: Michael J. Chamberlin". Molecular and Cell Biology. 2025-11-12. Retrieved 2025-11-24.
6. Sanchez, Alvaro; Osborne, Melisa L.; Friedman, Larry J.; Kondev, Jane; Gelles, Jeff (2011). "Mechanism of transcriptional repression at a bacterial promoter by analysis of single molecules". The EMBO Journal. 30 (19): 3940–3946. doi:10.1038/emboj.2011.273. PMC   3209775 . PMID   21829165.
7. Weitzmann, Carl J.; Cunningham, Philip R.; Ofengand, James (1990). "Cloning, in vitro transcription, and biological activity of Escherichia coli 23S ribosomal RNA". Nucleic Acids Research. 18 (12): 3515–3520. doi:10.1093/nar/18.12.3515. ISSN   0305-1048.
8. MacLellan, Shawn R.; Eiamphungporn, Warawan; Helmann, John D. (2008-10-21). "ROMA: An in vitro approach to defining target genes for transcription regulators". Methods. 47 (1): 73–77. doi:10.1016/j.ymeth.2008.10.009.
9. MacLellan, Shawn R.; Wecke, Tina; Helmann, John D. (2008-07-23). "A previously unidentified σ factor and two accessory proteins regulate oxalate decarboxylase expression in Bacillus subtilis". Molecular Microbiology. 69 (4): 954–967. doi:10.1111/j.1365-2958.2008.06331.x. ISSN   0950-382X.
10. Schmidt, Pascal; Brandt, David; Busche, Tobias; Kalinowski, Jörn (2023-05-25). "Characterization of Bacterial Transcriptional Regulatory Networks in Escherichia coli through Genome-Wide In Vitro Run-Off Transcription/RNA-seq (ROSE)". Microorganisms. 11 (6): 1388. doi:10.3390/microorganisms11061388. ISSN   2076-2607.
11. Chamberlin, Michael; Ring, Janet (1973-03-25). "Characterization of T7-specific Ribonucleic Acid Polymerase". Journal of Biological Chemistry. 248 (6): 2235–2244. doi:10.1016/S0021-9258(19)44211-7.
12. Lyakhov, Dmitry L; He, Biao; Zhang, Xing; Studier, F.William; Dunn, John J; McAllister, William T (1998-07-04). "Pausing and termination by bacteriophage T7 RNA polymerase". Journal of Molecular Biology. 280 (2): 201–213. doi:10.1006/jmbi.1998.1854.
13. Milligan, John F.; Groebe, Duncan R.; Witherell, Gary W.; Uhlenbeck, Olke C. (1987). "Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates". Nucleic Acids Research. 15 (21): 8783–8798. doi:10.1093/nar/15.21.8783. ISSN   0305-1048.
14. Melton, D.A.; Krieg, P.A.; Rebagliati, M.R.; Maniatis, T.; Zinn, K.; Green, M.R. (1984). "Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter". Nucleic Acids Research. 12 (18): 7035–7056. doi:10.1093/nar/12.18.7035. ISSN   0305-1048.
15. Zhu, Bin; Tabor, Stanley; Richardson, Charles C. (2014-03-01). "Syn5 RNA polymerase synthesizes precise run-off RNA products". Nucleic Acids Research. 42 (5): e33 –e33. doi:10.1093/nar/gkt1193. ISSN   0305-1048.