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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.
(This polymerase originates from the T7 phage, a bacteriophage virus which infects E. coli bacterial cells and is capable of integrating its DNA into the host DNA, as well as overriding its cellular machinery to produce more copies of itself.) T7 RNA polymerase is responsible for beginning transcription at the T7 promoter of the transformed vector.
The T7 promoter sequence is used extensively in molecular biology due to its extremely high affinity for T7 RNA polymerase and thus high level of expression. [3] [2] T7 has been used as a model in synthetic biology. Chan et al. (2005) "refactored" the genome of T7, replacing approximately 12 kbp of its genome with engineered DNA. [15]
Structure of eukaryotic RNA polymerase II (light blue) in complex with α-amanitin (red), a strong poison found in death cap mushrooms that targets this vital enzyme Eukaryotes have multiple types of nuclear RNAP, each responsible for synthesis of a distinct subset of RNA.
DE3 carries a T7 RNA polymerase (RNAP) gene under the control of a lacUV5 promoter (lacUV5-T7 gene 1). T7-RNAP is highly specific to the T7 promoter and orthogonal to native E. coli promoters. Therefore the T7-RNAP only transcribes (exogenously introduced) genes that are regulated by a T7 promoter. [6]
T7 RNA polymerase binds to the promoter region on the double strand. Since T7 RNA polymerase can only transcribe in the 3' to 5' direction [15] the sense DNA is transcribed and an anti-sense RNA is produced. This is repeated, and the polymerase continuously produces complementary RNA strands of this template which results in amplification.
A DNA polymerase; A suitable buffer that is compatible with the polymerase. A short DNA or RNA primer; A circular DNA template; Deoxynucleotide triphosphates (dNTPs) The detection methods of RCA product. The polymerases used in RCA are Phi29, Bst, and Vent exo-DNA polymerase for DNA amplification, and T7 RNA polymerase for RNA amplification ...
The Pribnow box has a function similar to the TATA box that occurs in promoters in eukaryotes and archaea: it is recognized and bound by a subunit of RNA polymerase during initiation of transcription. [3] This region of the DNA is also the first place where base pairs separate during prokaryotic transcription to allow access to the template strand.
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