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Expression Using the T7 RNA Polymerase/Promoter System

Expression Using the T7 RNA Polymerase/Promoter System This unit describes the expression of genes by placing them under the control of the bacteriophage T7 RNA polymerase. T7 RNA polymerase is a very active enzyme: it synthesizes RNA at a rate several times that of E. coli RNA polymerase and it terminates transcription less frequently; in fact, its transcription can circumnavigate a plasmid, resulting in RNA several times the plasmid length in size. T7 RNA polymerase is also highly selective for initiation at its own promoter sequences and is resistant to antibiotics such as rifampicin that inhibit E. coli RNA polymerase. Consequently, the addition of rifampicin to cells that are producing T7 RNA polymerase results in the exclusive expression of genes under the control of a T7 RNA polymerase promoter (pT7). In the , two plasmids are maintained within the same E. coli cell. One (the expression vector) contains pT7 upstream of the gene to be expressed. The second contains the T7 RNA polymerase gene under the control of a heat‐inducible E. coli promoter. Upon heat induction, the T7 RNA polymerase is produced and initiates transcription on the expression vector, resulting in turn in the expression of the gene(s) under the control of pT7. If desired, the gene products can be uniquely labeled by carrying out the procedure in minimal medium, adding rifampicin to inhibit the E. coli RNA polymerase, and then labeling the proteins with [35S]methionine. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Current Protocols in Molecular Biology Wiley

Expression Using the T7 RNA Polymerase/Promoter System

Tabor, Stanley
Current Protocols in Molecular Biology , Volume 11 (1) – Jul 1, 1990
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References (14)

Publisher
Wiley
Copyright
© John Wiley and Sons
ISSN
1934-3639
eISSN
1934-3647
DOI
10.1002/0471142727.mb1602s11
Publisher site
See Article on Publisher Site

Abstract

This unit describes the expression of genes by placing them under the control of the bacteriophage T7 RNA polymerase. T7 RNA polymerase is a very active enzyme: it synthesizes RNA at a rate several times that of E. coli RNA polymerase and it terminates transcription less frequently; in fact, its transcription can circumnavigate a plasmid, resulting in RNA several times the plasmid length in size. T7 RNA polymerase is also highly selective for initiation at its own promoter sequences and is resistant to antibiotics such as rifampicin that inhibit E. coli RNA polymerase. Consequently, the addition of rifampicin to cells that are producing T7 RNA polymerase results in the exclusive expression of genes under the control of a T7 RNA polymerase promoter (pT7). In the , two plasmids are maintained within the same E. coli cell. One (the expression vector) contains pT7 upstream of the gene to be expressed. The second contains the T7 RNA polymerase gene under the control of a heat‐inducible E. coli promoter. Upon heat induction, the T7 RNA polymerase is produced and initiates transcription on the expression vector, resulting in turn in the expression of the gene(s) under the control of pT7. If desired, the gene products can be uniquely labeled by carrying out the procedure in minimal medium, adding rifampicin to inhibit the E. coli RNA polymerase, and then labeling the proteins with [35S]methionine.

Journal

Current Protocols in Molecular Biology – Wiley

Published: Jul 1, 1990

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