Tagged RNA Polymerases and Method for Producing Them
Inventors: Robert Landick, Rachel Mooney
The Wisconsin Alumni Research Foundation (WARF) is seeking commercial partners interested in developing DNA constructs to create strains of E. coli that produce several different types of tagged RNA polymerases.
Much of the research on RNA transcription is focused on the action of RNA polymerases. Thus, improved methods for purifying and characterizing RNA polymerase greatly enhance studies of transcription. E. coli RNA polymerase is a multisubunit protein that consists in part of two large subunits, beta prime (155 kd) and beta (150 kd).
UW-Madison researchers have developed DNA constructs to create strains of E. coli (RL916 and RL1200) that produce several different types of tagged RNA polymerases. This technique yields E. coli strains that produce RNA polymerase with tags on the C-terminus of the beta prime subunit, a position that can be derivatized without adverse effects on enzyme activity. Biochemical tags that have been added include S. aureus protein A, E. coli biotin carrier protein, green fluorescent protein and beta galactosidase.
- The tagged RNA polymerases can be used in affinity purification, immobilized transcription, visualization of RNA polymerases in vivo and single molecule transcription.
- The modified RNA polymerases also can be immobilized and used in drug screening, such as screens for transcription inhibitors.
- The strains produce only the desired, modified RNA polymerase, greatly increasing yield and simplifying purification.