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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.
Usually, each member of this DNA library has a T7 RNA polymerase transcription site and a ribosomal binding site at the 5’ end. The T7 promoter region allows large-scale in vitro T7 transcription to transcribe the DNA library into an mRNA library, which provides templates for the in vitro translation reaction later.
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.
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]
Although this polypeptide has the same function as the three nuclear DNA-directed RNA polymerases, it is more closely related to RNA polymerases of bacteriophage (including T7 RNA polymerase), mitochondrial polymerases of lower eukaryotes as well as chloroplastic RpoT polymerases. [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.
Abortive initiation is a normal process of transcription and occurs both in vitro and in vivo. [2] After each nucleotide-addition step in initial transcription, RNA polymerase, stochastically, can proceed on the pathway toward promoter escape (productive initiation) or can release the RNA product and revert to the RNA polymerase-promoter open complex (abortive initiation).
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