T7 RNA Polymerase
(RNA Nucleotidyl transferase, E.C.2.7.7.6)
Source:
E. coli strain containing an overproducing clone of T7 RNA Polymerase(1)
Description:
T7 RNA Polymerase is a single-subunit enzyme produced by bacteriophage T7(2). It is highly specific for T7 promoter and terminator sequences(2,3). It has been widely used for the rapid synthesis in vitro of specific RNAs. These transcripts can be used directly as substrates for studies of RNA structure or metabolism. If the transcripts are suitably labeled, they can also be used as sensitive hybridization probes. T7 RNA Polymerase, along with chain-terminating nucleoside trihosphates(4), have also been used for the direct sequencing of DNA. T7 RNA Polymerase is also used to generate capped mRNA for expression studies in oocytes and other cells(5).
Properties:
Molecular Weight: 98.8 kDa
Optimum pH: 7.7 - 8.3
Optimum Temperature: 37°C
Requirement for Divalent Cation: Mg2+ (optimal concentration is 20mM)
Michaelis Constants(2): 40μM ATP, 160μM GTP, 60μM UTP, 80μM CTP
Purity:
Greater than 99% pure as determined by SDS-PAGE. Tested for contaminating nonspecific endonucleases, exonucleases and ribonucleases.
Storage Buffer:
20mM Tris-HCl, pH 7.5, 100mM NaCl, 0.1mM EDTA, 1mM DTT, 0.01% Triton®X-100, 50% glycerol.
Assay Conditions:
The reaction mixture (100 μl) contains 40mM Tris-HCl (pH 8.0), 20mM MgCl2, 5mM DTT, 400μM rNTP (A,C,G), 400μM radiolabeled UTP (30 cpm/pmol), 20 μg/ml T7 DNA template and 50 μg/ml BSA.
Unit Definition:
One unit is the amount of enzyme required to catalyze the incorporation of 1 nmol of labeled ribonucleoside triphosphate into acid insoluble material in 1 hr at 37°C, under standard assay conditions.
Concentration:
Standard Concentration (PN 70047Y): 20 units/μl
High Concentration (PN 70001Y/Z): > 80 units/μl
Tested User Friendly™ Functional Test:
Transcription from plasmid containing T7 promoter; >10 μg of RNA can be produced from 1 μg of supercoiled template.
PROTOCOL FOR IN VITRO TRANSCRIPTION WITH T7 RNA POLYMERASE:
1. For a 50 μl reaction add the following:
10X T7 RNA Polymerase Transcription Buffer 5 μl
Ribonuclease Inhibitor (PN 71571) 10 units
ATP, 10mM 5 μl
CTP, 10mM 5 μl
GTP, 10mM 5 μl
[α-33P]-UTP, 10mM 5 μl
Linearized DNA containing T7 promoter 2 μg
T7 RNA Polymerase 10 - 20 units
RNase-Free Water (PN 70783) to 50 μl
2. Incubate at 37°C for 1-2 hours.
3. Stop reaction by adding 1 μl of 0.5M EDTA or by incubating at 75°C for 10 min.
Note: Either [α-32P]-UTP or [α-33P]-UTP may be used.
Functionally Tested 10X T7 RNA Polymerase Transcription Buffer (1 ml included):
400mM Tris-HCl (pH 8.0), 150mM MgCl2 , 50mM DTT.
References:
- TABOR, S. AND RICHARDSON, C. C. (1985) Proc. Natl. Acad. Sci. USA 82,
1074-1078. - CHAMBERLIN, M. AND RING J. (1973) J. Biol. Chem. 248, 2235-2244, 2245-2250.
- CHAMBERLIN, M. AND RYAN, T. (1982) in The Enzymes, 3rd edition, ed. P. D. Boyer (Academic Press, New York.) 15, 87-108.
- AXELROD, V. D. AND KRAMER, F. R.(1985) Biochemistry 24, 5716-5723.
- SAMBROOK, J. AND RUSSELL, D. W. (2001) Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor, New York, 9.87-9.88.
- AUSUBEL, F. M., BRENT, R., KINGSTON, R. E., MOORE, D. D., SEIDMAN, J. G., SMITH, J. A. AND STRUHL, K. (1998) Current Protocols in Molecular Biology (John Wiley and Sons, Inc.).
Applications:
- Production of RNA transcripts for hybridization probes.
- Production of large amounts of discrete sized RNA.
- Production of capped or modified RNA transcripts.
Storage:
Shipped on dry ice. Store at -20°C.