Open AccessCloning and expression of the gene for bacteriophage T7 RNA polymerase.
Author(s) -
Parichehre Davanloo,
Alan H. Rosenberg,
John J. Dunn,
F. William Studier
Publication year - 1984
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.81.7.2035
Subject(s) - t7 rna polymerase , rna polymerase , biology , microbiology and biotechnology , polymerase , rna dependent rna polymerase , gene , bacteriophage , rna polymerase i , sigma factor , promoter , rna , gene expression , escherichia coli , genetics
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.