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Run-off Transcription

A run-off transcription assay can be conducted in vitro to identify the position of the transcription start site (+1) 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 To perform a run-off transcription assay, a gene of interest, along with its 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.1,4 DNA needs to be highly purified prior to running this assay.1 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 product mRNA produced.4

Fig 1. Run-off Transcription Assay. Cartooned is part of a plasmid that contains (A) a promoter and downstream DNA with a known restriction enzyme site. (B) The DNA is cut with the restriction enzyme, leaving DNA fragment (in this case 328 bp from the +1). (C) Transcription is initiated by the addition of radiolabeled UTP, NTPs and RNA Polymerase II. (D) RNA Polymerase II continues with transcription until it reaches the end of the DNA fragment, where it then falls off, or “runs off”, resulting in. an incomplete mRNA of a defined length. (E) The RNA is separate by gel electrophoresis, along with a size standards, and detected by autoradiography to determine the length and intensity of the mRNA. References: Loewenstein PM, Song C, Green M. The Use of In Vitro Transcription to Probe Regulatory Functions of Viral Protein Domains. Adenovirus Methods and Protocols. 2007;2:15-31. Run-Off Transcription. Available at: http://molecularstation.com/dna/run-off-transcription. Accessed April 16, 2014. Lelandais C, Gutierres S, Mathieu C, et al. 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 Res. 1996;24:4798-4804. Transcription in eukaryotes. In: Allison LA. Fundamental Molecular Biology. 2011:318. Available at: http://www.blackwellpublishing.com/allison/docs/sample_ch11.pdf. Accessed April 18, 2014.