Cloning and expression of the gene for bacteriophage T7 RNA polymerase

Journal Article · Sun Apr 01 04:00:00 EST 1984 · Proc. Natl. Acad. Sci. U.S.A.; (United States)

DOI:https://doi.org/10.1073/pnas.81.7.2035· OSTI ID:5932644

Davanloo, P; Rosenberg, A H; Dunn, J J; Studier, F W

The complete coding sequence of the gene for bacteriophage T7 RNA polymerase (T7 gene 1) has been cloned in the plasmid pBR322. Large amounts of active enzyme can be accumulated in Escherichia coli when the cloned gene is transcribed from the lac UV5 promoter. A protease activity that apparently can nick the protein without causing it to fall apart can be a problem during purification, but a procedure is described that gives good yields of essentially homogeneous, highly active enzyme suitable for biochemical and physical studies. T7 RNA polymerase has a stringent specificity for its own promoters and will selectively transcribe DNA that has been linked to such a promoter. This specificity makes the enzyme useful both for producing specific RNAs in vitro and for directing the expression of selected genes inside the cell. Having the cloned gene also makes possible a detailed mutational analysis of the functioning of T7 RNA polymerase. 25 references, 3 figures.

• Research Organization: Brookhaven National Lab., Upton, NY
• OSTI ID: 5932644
• Journal Information: Proc. Natl. Acad. Sci. U.S.A.; (United States), Journal Name: Proc. Natl. Acad. Sci. U.S.A.; (United States) Vol. 81; ISSN PNASA
• Country of Publication: United States
• Language: English

Related Subjects

550400* -- Genetics
59 BASIC BIOLOGICAL SCIENCES
BACTERIA
BACTERIOPHAGES
CLONING
DNA
DNA SEQUENCING
ENZYME ACTIVITY
ENZYMES
ESCHERICHIA COLI
GENES
MICROORGANISMS
NUCLEIC ACIDS
NUCLEOTIDYLTRANSFERASES
ORGANIC COMPOUNDS
PARASITES
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
PURIFICATION
RNA POLYMERASES
STRUCTURAL CHEMICAL ANALYSIS
TRANSFERASES
VIRUSES