T7 RNA polymerase

T7 RNA Polymerase (blue) producing mRNA (light-blue) from a double-stranded DNA template (orange).

T7 RNA Polymerase is an RNA polymerase from the T7 bacteriophage that catalyzes the formation of RNA in the 5'→ 3' direction.

Activity

T7 polymerase is extremely promoter-specific and transcribes only DNA downstream of a T7 promoter (TAATACGACTCACTATAG, transcription beginning with the 3' G). 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.

Structure

T7 polymerase has been crystallised in several forms and the structures placed in the PDB. The different structures are listed here. These explain how T7 polymerase binds to DNA and transcribes it.

Related proteins

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.

Application

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.

Sources

• - note that the nuclease activity reported here is an artifact.

External links

• T7 RNA Polymerase Enzymology
• OpenWetWare