Bacteriophage T7 RNA polymerase

Na salts of ribonucleotide triphosphates (Roche or Sigma) bovine serum albumin RNase-free, 20 mg/ml (Roche) RNasin ribonuclease inhibitor, 40 U/ml (Promega) both bacteriophage T7 RNA polymerase and RNA Cap structure analog m7G(5/)ppp(5/)G are from BioLabs DNase-RNase-free (Roche) complete EDTA-free proteinase inhibitors cocktail (Roche) pyruvate kinase (PK) (Roche). 

Figure 6. Amplification of RNA molecules by assays that are sequence- insensitive. The first assay (upper part) combines the polymerase chain reaction (PCR) of DNA templates with reverse transcription and transcription. Commonly used enzymes are TAQ-polym-erase, HIV reverse transcriptase and bacteriophage T7 RNA polymerase. The assay requires a temperature program applying higher temperatures for double strand dissociation. The second assay (lower part) shows the self-sustained sequence replication reaction (3SR) which can be carried out isothermally because double strand dissociation is replaced by enzymatic digestion of the RNA strand in the RNA-DNA duplex. The enzymes used are HIV reverse transcriptase, RNase H and T7 RNA polymerase.

Studier, F. W. and Moffatt, B. A. (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189, 113-130. 

Genetic Selection of an Error-prone Variant of Bacteriophage T7 RNA Polymerase... 

Stahl, S., and Zinn, K. (1981). Nucleotide Sequence of the Cloned Gene for Bacteriophage T7 RNA Polymerase. J Mol Biol 148 481. 

Tabor, S. and Richardson, C. C (1985) A bacteriophage T7 RNA polymerase/pro-moter system for controlled exclusive expression of specific genes. Proc Natl Acad Sa 82,1074-1078. 

Dimitti, A., Bums, J.A., Broyde, S., and Sdcchitano, D.A. (2008) Transcription elongation past 06-methylguanine by human RNA polymerase II and bacteriophage T7 RNA polymerase. Nucleic Adds Res., 36, 6459-6471. 

Bacteriophage SP6 RNA polymerase is blocked at single-strand breaks that form at an abasic site where the 2 -deoxyribose is present on either the 3 - or 5 -side of the nick, but bacteriophage T7 RNA polymerase bypasses abasic strand breaks... 

Choi, D.J., Marino-Alessandri, D.J., Geacintov, N.E., and Scicchitano, D.A. (1994) Site-specific benzo[a]pyrene diol epoxide-DNA adducts inhibit transcription elongation by bacteriophage T7 RNA polymerase. Biochemistry, 33, 780-787. 

The desC genes from Synechocystis sp. PCC 6803 and A. variahilis were introduced into pET-3a and overexpressed in E. coli under the control of bacteriophage T7 RNA polymerase. The cells were supplied with 18 0, which is present at very low levels in E. coli cells under normal growth conditions. The externally supplied 18 0 was esterified at both the sn-1 and sn-2 positions of the glycerol moiety of phosphatidylglycerol and phosphatidyl-ethanolamine. The overexpressed A9 acyl-lipid desaturase of Synechocystis sp. PCC 6803 and of A. variahilis was active in converting 18 0 to 18 1(9) at the sn-1 position but not at the sn-2 position, and it did not desaturate 16 0 at the sn-1 or the sn-2 position. These observations lead us to conclude that the A9 acyl-lipid desaturases from Synechocystis sp. PCC 6803 and A. variahilis are specific to 18 0 and the sn-1 position but are nonspecific with respect to the head group. 

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