Cytoplasmic expression system based on constitutive synthesis of bacteriophage T7 RNA polymerase in mammalian cells.

Cytoplasmic expression system based on constitutive synthesis of bacteriophage T7 RNA polymerase in mammalian cells.

A mouse cell line that constitutively synthesizes the bacteriophage T7 RNA polymerase was constructed. Fluorescence microscopy indicated that the T7 RNA polymerase was present in the cytoplasmic compartment. The system provided, therefore, a unique opportunity to study structural elements of mRNA that affect stability and translation. The in vivo activity of the bacteriophage polymerase was dem...

Bacteriophage T7 RNA polymerase-based expression in Pichia pastoris.

A novel Pichia pastoris expression vector (pEZT7) for the production of recombinant proteins employing prokaryotic bacteriophage T7 RNA polymerase (T7 RNAP) (EC 2.7.7.6) and the corresponding promoter pT7 was constructed. The gene for T7 RNAP was stably introduced into the P. pastoris chromosome 2 under control of the (endogenous) constitutive P. pastoris glyceraldehyde-3-phosphate dehydrogenas...

Expression of bacteriophage T7 RNA polymerase in avian and mammalian cells by a recombinant fowlpox virus.

The bacteriophage T7 RNA polymerase gene was integrated into the fowlpox virus genome under the control of the vaccinia virus early/late promoter, P7.5. The recombinant fowlpox virus, fpEFLT7pol, stably expressed T7 RNA polymerase in avian and mammalian cells, allowing transient expression of transfected genes under the control of the T7 promoter. The recombinant fowlpox virus expressing T7 RNA...

Enzymatic RNA Synthesis using Bacteriophage T7 RNA Polymerase

Kinetic mechanism of GTP binding and RNA synthesis during transcription initiation by bacteriophage T7 RNA polymerase.

We have used stopped-flow and rapid chemical quench-flow methods to investigate the kinetics of the early steps during transcription initiation by bacteriophage T7 RNA polymerase. Most promoters of T7 RNA polymerase initiate with two GTPs. The kinetics of GTP binding was investigated by monitoring the fluorescence changes resulting from GTP binding to polymerase and fluorescent 2-aminopurine-co...