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| T7 RNA polymerase | |||||||
|---|---|---|---|---|---|---|---|
 T7 RNA Polymerase (blue) producing mRNA (light-blue) from a double-stranded DNA template (orange). | |||||||
| Identifiers | |||||||
| Organism | T7 phage | ||||||
| Symbol | 1 | ||||||
| PDB | 1MSW | ||||||
| UniProt | P00573 | ||||||
| |||||||
T7 RNA Polymerase is anRNA polymerase from theT7bacteriophage that catalyzes the formation ofRNA from DNA in the 5'→ 3' direction.[1]
T7 polymerase is extremelypromoter-specific and transcribes only DNA downstream of a T7 promoter.[2] The T7 polymerase also requires a double stranded 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.
The promoter is recognized for binding and initiation of the transcription. The consensus in T7 and related phages is:[2]
5' * 3'T7 TAATACGACTCACTATAGGGAGAT3 AATTAACCCTCACTAAAGGGAGAK11 AATTAGGGCACACTATAGGGAGASP6 ATTTACGACACACTATAGAAGAA bind------------ -----------init
Transcription begins at the asterisk-marked guanine.[2]
T7 polymerase has been crystallised in several forms and the structures placed in thePDB. These explain how T7 polymerase binds to DNA and transcribes it. The N-terminal domain moves around as the elongation complex forms. The ssRNAP holds a DNA-RNA hybrid of 8bp.[3] Abeta-hairpin specificity loop (residues 739-770 in T7) recognizes the promoter; swapping it out for one found in T3 RNAP makes the polymerase recognize T3 promoters instead.[2]
Similar to otherviral nucleic acid polymerases, includingT7 DNA polymerase from the same phage, the conserved C-terminal of T7 ssRNAP employs a fold whose organization has been likened to the shape of a right hand with three subdomains termed fingers, palm, and thumb.[4] The N-terminal is less conserved. It forms a promoter-binding domain (PBD) with helix bundles in phage ssRNAPs,[5] a feature not found in mitochondrial ssRNAPs.[6]
| DNA-directed RNA polymerase, phage-type | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Identifiers | |||||||||
| Symbol | RNA_pol | ||||||||
| Pfam | PF00940 | ||||||||
| InterPro | IPR002092 | ||||||||
| SCOP2 | 1msw /SCOPe /SUPFAM | ||||||||
| |||||||||
T7 polymerase is a representative member of thesingle-subunit DNA-dependent RNAP (ssRNAP) family. Other members include phage T3 and SP6 RNA polymerases, the mitochondrial RNA polymerase (POLRMT), and the chloroplastic ssRNAP.[7][8] The ssRNAP family 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 antibioticrifampicin. This family is related to single-subunitreverse transcriptase andDNA polymerase.[9]
In biotechnology applications, T7 RNA polymerase is commonly used to transcribe DNA that has been cloned intovectors 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 byspermidine andin vitro activity is increased by the presence of carrier proteins (such asBSA).[10][11]
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.
T7 RNA polymerase is used in the synthesis of mRNA and sgRNA.[12]