A run-off transcription assay 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]
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).[5]
References
- ^ a b c d e f g h Loewenstein, P. M.; Song, C. Z.; Green, M (2007). The use of in vitro transcription to probe regulatory functions of viral protein domains. 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.
- ^ a b c d e f g h "Run-off Transcription". Molecular Station. Archived from the original on April 22, 2014. Retrieved April 16, 2014.
- ^ 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.
- ^ a b c d e f Allison, Lizabeth. "Fundamental molecular biology, chapter 11" (PDF). BlackWell Publishing. Retrieved April 18, 2014.
- ^ 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.