| 词条 | T7 RNA polymerase |
| 释义 |
|image=T7 RNA polymerase.jpg |caption=T7 RNA Polymerase (blue) producing mRNA (light-blue) from a double-stranded DNA template (orange). |UniProt=P00573 |PDB=1MSW }} T7 RNA Polymerase is an RNA polymerase from the T7 bacteriophage that catalyzes the formation of RNA from DNA in the 5'→ 3' direction. ActivityT7 polymerase is extremely promoter-specific and transcribes only DNA downstream of a T7 promoter.[1] 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. PromoterThe promoter is recognized for binding and initiation of the transcription. The consensus in T7 and related pgages is:[1] 5’ * 3‘ T7 TAATACGACTCACTATAGGGAGA T3 AATTAACCCTCACTAAAGGGAGA K11 AATTAGGGCACACTATAGGGAGA SP6 ATTTACGACACACTATAGAAGAA bind------------ -----------init Transcription begins at the asterisk-marked guanine.[1] StructureT7 polymerase has been crystallised in several forms and the structures placed in the PDB. 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.[1] A beta-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 other viral nucleic acid polymerases, including T7 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.[3] The N-terminal is less conserved. It forms a promoter-binding domain (PBD) with helix bundles in phage ssRNAPs,[4] a feature not found in mitochondrial ssRNAPs.[5] Related proteins{{Pfam box|Pfam=PF00940|Symbol=RNA_pol|Name=DNA-directed RNA polymerase, phage-type|InterPro=IPR002092|SCOP=1msw}}T7 polymerase is a representative member of the single-subunit DNA-dependent RNAP (ssRNAP) family. Other members include phage T3 and SP6 RNA polymerases, the mitochondrial RNA polymerase (POLRMT), and the chloroplastic ssRNAP.[6][7] 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 antibiotic rifampicin. This family is related to single-subunit reverse transcriptase and DNA polymerase.[8] ApplicationIn 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 spermidine and in vitro activity is increased by the presence of carrier proteins (such as BSA)[9][10] 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. References1. ^{{cite journal | vauthors = Tahirov TH, Temiakov D, Anikin M, Patlan V, McAllister WT, Vassylyev DG, Yokoyama S | title = Structure of a T7 RNA polymerase elongation complex at 2.9 A resolution | journal = Nature | volume = 420 | issue = 6911 | pages = 43–50 | date = November 2002 | pmid = 12422209 | doi = 10.1038/nature01129 }} 2. ^1 2 3 {{cite journal | vauthors = Rong M, He B, McAllister WT, Durbin RK | title = Promoter specificity determinants of T7 RNA polymerase | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 95 | issue = 2 | pages = 515–9 | date = January 1998 | pmid = 9435223 | pmc = 1845 | doi = 10.1073/pnas.95.2.515 }} 3. ^{{cite journal | vauthors = Hansen JL, Long AM, Schultz SC | title = Structure of the RNA-dependent RNA polymerase of poliovirus | journal = Structure | volume = 5 | issue = 8 | pages = 1109–22 | date = August 1997 | pmid = 9309225 | doi = 10.1016/S0969-2126(97)00261-X }} 4. ^{{cite journal | vauthors = Durniak KJ, Bailey S, Steitz TA | title = The structure of a transcribing T7 RNA polymerase in transition from initiation to elongation | journal = Science | volume = 322 | issue = 5901 | pages = 553–7 | date = October 2008 | pmid = 18948533 | pmc = 2892258 | doi = 10.1126/science.1163433 }} 5. ^{{cite journal | vauthors = Hillen HS, Morozov YI, Sarfallah A, Temiakov D, Cramer P | title = Structural Basis of Mitochondrial Transcription Initiation | journal = Cell | volume = 171 | issue = 5 | pages = 1072–1081.e10 | date = November 2017 | pmid = 29149603 | doi = 10.1016/j.cell.2017.10.036 }} 6. ^{{cite journal | vauthors = McAllister WT, Raskin CA | title = The phage RNA polymerases are related to DNA polymerases and reverse transcriptases | journal = Molecular Microbiology | volume = 10 | issue = 1 | pages = 1–6 | date = October 1993 | pmid = 7526118 | doi = 10.1111/j.1365-2958.1993.tb00897.x }} 7. ^{{cite journal | vauthors = Hedtke B, Börner T, Weihe A | title = Mitochondrial and chloroplast phage-type RNA polymerases in Arabidopsis | journal = Science | volume = 277 | issue = 5327 | pages = 809–11 | date = August 1997 | pmid = 9242608 | doi = 10.1126/science.277.5327.809 }} 8. ^{{cite journal | vauthors = Cermakian N, Ikeda TM, Miramontes P, Lang BF, Gray MW, Cedergren R | title = On the evolution of the single-subunit RNA polymerases | journal = Journal of Molecular Evolution | volume = 45 | issue = 6 | pages = 671–81 | date = December 1997 | pmid = 9419244 | url = https://www.researchgate.net/publication/13811504 | doi = 10.1007/PL00006271 }} 9. ^{{cite journal | vauthors = Chamberlin M, Ring J | title = Characterization of T7-specific ribonucleic acid polymerase. 1. General properties of the enzymatic reaction and the template specificity of the enzyme | journal = The Journal of Biological Chemistry | volume = 248 | issue = 6 | pages = 2235–44 | date = March 1973 | pmid = 4570474 | doi = | url = }} 10. ^{{cite journal | vauthors = Maslak M, Martin CT | title = Effects of solution conditions on the steady-state kinetics of initiation of transcription by T7 RNA polymerase | journal = Biochemistry | volume = 33 | issue = 22 | pages = 6918–24 | date = June 1994 | pmid = 7911327 | doi = 10.1021/bi00188a022| url = }} Further reading{{refbegin}}
External links
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