Cloning and expression of the gene for bacteriophage T7 RNA polymerase.

Abstract

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.Bacteriophage T7 RNA polymerase (EC 2.7.7.6) is produced early in T7 infection and plays a central role in regulating gene expression (for a review, see ref. 1). A single-chain enzyme with a molecular weight close to 100,000, T7 RNA polymerase has a stringent specificity for its own promoters, which contain a highly conserved sequence of 23 continuous base pairs including the start site for the RNA. Seventeen such promoters are found in T7 DNA, but few if any such promoters are found in host DNA or in any other DNAs unrelated to T7. Once T7 RNA polymerase has been produced during infection, other T7 gene products inactivate the host RNA polymerase, leaving all transcription in the cell directed to T7 DNA.The great specificity of T7 RNA polymerase for its own promoters is not only central to the strategy of T7 infection but is also interesting and potentially useful to biochemists. Understanding the basis for this specificity is a challenge in its own right. Furthermore, the purified enzyme can be used to produce large amounts of specific RNAs simply by transcribing DNA that has been joined to a promoter for T7 RNA polymerase. Such RNAs could be useful as hybridization probes, mRNAs for in vitro translation, substrates for analyzing processing reactions or RNA splicing, or for any purpose requiring a specific RNA. T7 RNA polymerase, together with a suitably positioned promoter, can also be used to direct the transcription of selected genes inside the cell. Target genes potentially could be expressed at very high levels, using the same strategy employed by T7 itself: once T7 RNA polymerase had been made, the host RNA polymerase could be inactivated, thereby eliminating the synthesis of competing mRNAs. The T7 RNA polymerase made during T7 infection has already been shown to be capable of directing the transcription of genes cloned in plasmids (2-4).The yield of purified T7 RNA polymerase from infected cells is not particularly good, because the enzyme is synthesized for only a few minutes during infection and does not accumulate to high levels. Nor does T7 infection provide an ...

Solution Structure of Domain 6 from a Self‐Splicing Group II Intron Ribozyme: A Mg2+ Binding Site is Located Close to the Stacked Branch Adenosine

“…T7 polymerase used for in vitro transcription was homemade. [20,42] The All chemicals used were at least puriss p.a. and purchased from either Fluka-Sigma-Aldrich, Buchs (Switzerland) or Brunschwig Chemie, Amsterdam (The Netherlands).…”

Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site

“…Unlabeled nucleoside triphosphates were purchased from P-L Biochemicals, guanosine was from Sigma, [y3*P]ATP was from ICN Biomedicals or New England Nuclear, T4 polynucleotide kinase was from United States Biochemicals, calf intestinal phosphatase was from New England Nuclear, RNase T1 was from Sankyo, and ScaI restriction endonuclease was from New England Biolabs. Phage T7 RNA polymerase was isolated from Escherichia coli strain BL2 1, containing the plasmid PAR 12 1 9 (Davanloo et al, 1984), by A. Zaug.…”

5S rRNA sugar‐phosphate backbone protection in complexes with specific ribosomal proteins

“…The T7 RNA polymerase was isolated from £ eoli strain BL21 containing the plasmid pAR 1219 according to a published procedure [9]. Individual ribosomal proteins L5, L18 and L25 were obtained as described in [10].…”