In vitro transcription

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The in vitro transcription , so the DNA -dependent synthesis of RNA in vitro, is a molecular biological method for the production of RNA, and for the investigation of promoters and their activation by transcription factors .

Synthesis of RNA

RNA generated in vitro is used to study RNA folding and RNA stability. It is also as a template ( template , English template ) for the In-vitro -Translation or expression in oocytes needed. Protein synthesis is also carried out as coupled transcription / translation. An important application is the production of labeled RNA probes, probes (engl., Also riboprobes riboprobe ) can be mentioned. In this case, transcripts aresynthesizedwith radioactively ( 32 P, 35 S) or fluorescence- labeled ribonucleotides . Alternatively, the transcription for indirect detection is u. a. performed with nucleotidesmodifiedwith biotin or digoxigenin . For these purposes,DNA templates generatedwith the polymerase chain reaction (PCR) are cloned into suitable plasmid vectors , such as pGEM-T Easy , which have bacteriophage SP6, T3 or T7promotersflanking the multiple cloning site . The plasmid is thenlinearizedat the 3 'end with a restriction enzyme and treated with the appropriate RNA polymerase , e.g. B. T7 RNA polymerase , and the appropriate ribonucleotides to generate run-off transcripts. In this way sense and antisense samples can be generated. As an alternative to cloning, PCR fragments can also be used directly if one of the primers used contains a phage promoter. This possibility is important for the hybridization of DNA microarrays .

Study of promoters

The in vitro investigation of the promoters of cloned genes in extracts and protein fractions allows conclusions to be drawn about the DNA elements involved as well as the proteins involved in the transcription. The eukaryotic RNA polymerases require additional proteins, the basic transcription factors and the specific activators and repressors for initiation and elongation . The American molecular biologist Robert G. Roeder made a significant contribution to understanding the regulation of gene activity with in vitro transcription assays.

For a typical experiment, may require the DNA template, a promoter and to be transcribed region, upstream regulatory elements such as enhancers and silencers , a cell extract , of all the proteins necessary for transcription contains the four ribonucleotides and a suitable buffer with the necessary ions. If you want to examine the cis elements, mutate or delete regulatory areas, if you want to examine trans elements, use protein fractions that lack certain proteins. Accordingly, one can also examine the influence of recombinant proteins by adding them.

The transcription is usually detected by incorporating radioactive nucleotides, mostly [α- 32 P] - UTP . Either linearized or G-free templates are used. In the first case, run-off transcripts arise, while in the second case a template is used which has a section without guanine residues in the direction of transcription. Instead of GTP , 3'-O-methyl- GTP is used, the incorporation of which leads to a strand break. The transcripts are detected by autoradiography after polyacrylamide gel electrophoresis . The transcriptional activity can be determined by measuring the radioactivity of the specific gel band in a scintillation counter or with a phosphoimager .

literature

Individual evidence

  1. ↑ PA Krieg, DA Melton: Functional messenger RNAs are produced by SP6 in vitrotranscription of cloned cDNAs . In: Nucleic Acids Research . tape 12 , no. 18 , 1984, ISSN  0305-1048 , pp. 7057-7070 , doi : 10.1093 / nar / 12.18.7057 .
  2. ↑ DA Melton, PA Krieg, MR Rebagliati, T. Maniatis, K. Zinn, MR Green: Efficientin vitrosynthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter . In: Nucleic Acids Research . tape 12 , no. 18 , 1984, ISSN  0305-1048 , pp. 7035-7056 , doi : 10.1093 / nar / 12.18.7035 .
  3. ↑ John D. Dignam, Paul L. Martin, Barkur S. Shastry, Robert G. Roeder: [36] Eukaryotic gene transcription with purified components . In: Methods in Enzymology . tape 101 , 1983, ISSN  0076-6879 , pp. 582-598 , doi : 10.1016 / 0076-6879 (83) 01039-3 .
  4. ^ Robert G. Roeder: Transcriptional regulation and the role of diverse coactivators in animal cells . In: FEBS Letters . tape 579 , no. 4 , 2005, ISSN  0014-5793 , p. 909-915 , doi : 10.1016 / j.febslet.2004.12.007 .
  5. ↑ JD Dignam, RM Lebovitz, RG Roeder: Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei . In: Nucleic Acids Research . tape 11 , no. 5 , 1983, ISSN  0305-1048 , pp. 1475–1489 , doi : 10.1093 / nar / 11.5.1475 .
  6. ^ Jo C Wang, Michèle Sawadogo, Michael W Van Dyke: Plasmids for the in vitro analysis of RNA polymerase II-dependent transcription based on a G-free template . In: Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression . tape 1397 , no. 2 , 1998, ISSN  0167-4781 , p. 141-145 , doi : 10.1016 / S0167-4781 (98) 00012-8 .