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

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.

External links

Transferases: phosphorus-containing groups (EC 2.7)

2.7.1-2.7.4:
phosphotransferase/kinase
(PO₄)

2.7.1: OH acceptor	Hexo- Gluco- Fructo- Hepatic Galacto- Phosphofructo- 1 Liver Muscle Platelet 2 Riboflavin Shikimate Thymidine ADP-thymidine NAD⁺ Glycerol Pantothenate Mevalonate Pyruvate Deoxycytidine PFP Diacylglycerol Phosphoinositide 3 Class I PI 3 Class II PI 3 Sphingosine Glucose-1,6-bisphosphate synthase

2.7.2: COOH acceptor	Phosphoglycerate Aspartate kinase

2.7.3: N acceptor	Creatine

2.7.4: PO₄ acceptor	Phosphomevalonate Adenylate Nucleoside-diphosphate Uridylate Guanylate Thiamine-diphosphate

2.7.6: diphosphotransferase
(P₂O₇)

2.7.7: nucleotidyltransferase
(PO₄-nucleoside)

Polymerase

DNA polymerase	DNA-directed DNA polymerase I II III IV V RNA-directed DNA polymerase Reverse transcriptase Telomerase DNA nucleotidylexotransferase/Terminal deoxynucleotidyl transferase

RNA nucleotidyltransferase	RNA polymerase/DNA-directed RNA polymerase RNA polymerase I RNA polymerase II RNA polymerase III RNA polymerase IV Primase RNA-dependent RNA polymerase PNPase

Phosphorolytic
3' to 5' exoribonuclease

Nucleotidyltransferase

Guanylyltransferase

mRNA capping enzyme

Other

2.7.8: miscellaneous

Phosphatidyltransferases	CDP-diacylglycerol—glycerol-3-phosphate 3-phosphatidyltransferase CDP-diacylglycerol—serine O-phosphatidyltransferase CDP-diacylglycerol—inositol 3-phosphatidyltransferase CDP-diacylglycerol—choline O-phosphatidyltransferase

Glycosyl-1-phosphotransferase	N-acetylglucosamine-1-phosphate transferase

2.7.10-2.7.13: protein kinase
(PO₄; protein acceptor)

2.7.10: protein-tyrosine	see tyrosine kinases

2.7.11: protein-serine/threonine	see serine/threonine-specific protein kinases

2.7.12: protein-dual-specificity	see serine/threonine-specific protein kinases

2.7.13: protein-histidine	Protein-histidine pros-kinase Protein-histidine tele-kinase Histidine kinase

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