Construction and Properties of Plasmid Vectors Containing the trp Regulatory Region Suitable for Expressing Foreign Genes
The E. coli tryptophan (trp) operon consists of a regulatory region foIldwid by five structural genes (trpE through trpA)1,2. The trp structural gene products, Which are co-ordinately synthesized in equimolar amounts, catalyze the conversion of chorismate to tryptophan1,3. Transcription of the operon is repressed by tryptophan by two mechanisms. Tryptophan binds trp repressor (trpR) resulting in an increase in the affinity of the repressor for the trp operator4. Since the trp operator sequence overlaps the trp promoter sequence (see Fig. 1) binding of repressor prevents binding of RNA polymerase. Secondly, in the presence of tryptophan about 90% of the RNA polymerase molecules which are able to initiate, terminate transcription about 140 base pairs (bp) frcm the transcription start (see Fig. 1). The ability of the transcription terminator to function is determined by the level of tryptophanyl tRNA9.
KeywordsCold Spring Harbor Foreign Gene Multicopy Plasmid Transcription Terminator ClaI Site
Unable to display preview. Download preview PDF.
- 10.R. Grantham, C. Gautier, M. Gouy, R. Mercier, and A. Pavez, Codon catalog usage and the gencme hypothesis. Nucleic Acids Res. 8: 49 (1980).Google Scholar
- 12.J.H. Miller, in “Experiments in Molecular Genetics, Appendix I, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1972) Google Scholar
- 15.J.C. Edman, R.A. Hallewell, P. Valenzuela, H.M. Goodman, and W.J. Rutter, The synthesis of hepatitis B surface and core antigens in E. coli, Nature, in press.Google Scholar
- 22.D.V. Goeddel, H.M. Shepard, E. Yelverton, D. Leung, and R. Créa, Synthesis of human fibroblast interferon by E. coli, Nucleic Acids Res. 8: 4–57 (1980).Google Scholar
- 23.J.A. Martial, R.A. Hallewell, J.D. Baxter, and H.M. Goodman, Human growth hormone: complementary DNA cloning and expression in bacteria, Science 2ø5: 6–2 (1979).Google Scholar
- 28.C.K. Singleton, W.D. Roeder, G. Bogosian, R.L. Scmerville, and H.L. Weith, DNA sequence of the E. coli trpR gene and prediction of the amino acid sequence of Trp repressor, Nucleic Acids Res., in press.Google Scholar
- 31.W. Tacon, N. Carey, and S. Emtage, The construction and characterization of plasmid vectors suitable for the expression of all DNA phases under the control of the E. coli tryptophan prcmoter Molec. gen. Genet. 177: 427 (198ø).Google Scholar
- 33.G.E. Christie and T. Platt, A functional hybrid ribosome binding site in tryptophan operon mRNA of E. coli, J. Mol. Biol., in press.Google Scholar