T7 RNA polymerase
T7 polymerase is extremely promoter-specific and transcribes only DNA downstream of a T7 promoter. The T7 polymerase also requires a DNA template and Mg2+ ion as cofactor for the synthesis of RNA. It has a very low error rate. T7 polymerase has a molecular weight of 99 kDa.
Related family members include phage T3 and SP6 RNA polymerases, but this family is also related to the mitochondrial RNA polymerase. The T7 family of RNA polymerases is structurally and evolutionarily distinct from the multi-subunit family of RNA polymerases (including bacterial and eukaryotic sub-families). In contrast to bacterial RNA polymerases, T7 polymerase is not inhibited by the antibiotic rifampicin. Nevertheless, many common functional features are shared with these more complex enzymes.
In biotechnology applications, T7 RNA polymerase is commonly used to transcribe DNA that has been cloned into vectors that have two (different) phage promoters (e.g., T7 and T3, or T7 and SP6) in opposite orientation. RNA can be selectively synthesized from either strand of the insert DNA with the different polymerases. The enzyme is stimulated by BSA or spermidine.
Homogeneously labeled single-stranded RNA can be generated with this system. Transcripts can be non-radioactively labeled to high specific activity with certain labeled nucleotides.
- Martin CT, Esposito EA, Theis K, Gong P (2005). "Structure and function in promoter escape by T7 RNA polymerase". Prog. Nucleic Acid Res. Mol. Biol. 80: 323–47. doi:10.1016/S0079-6603(05)80008-X. PMID 16164978.
- Sousa R, Mukherjee S (2003). "T7 RNA polymerase". Prog. Nucleic Acid Res. Mol. Biol. 73: 1–41. doi:10.1016/S0079-6603(03)01001-8. PMID 12882513.
- McAllister WT (1993). "Structure and function of the bacteriophage T7 RNA polymerase (or, the virtues of simplicity)". Cell. Mol. Biol. Res. 39 (4): 385–91. PMID 8312975.
- Sastry SS, Ross BM (March 1997). "Nuclease activity of T7 RNA polymerase and the heterogeneity of transcription elongation complexes". J. Biol. Chem. 272 (13): 8644–52. doi:10.1074/jbc.272.13.8644. PMID 9079696. - note that the nuclease activity reported here is an artifact.
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