Abstract
A system is described which allows testing of specific protein-DNA-interactions. The system consists of two mutually compatible plasmids carrying different origins of replication and resistance markers. One plasmid carries a lac I gene in which the DNA-recognizing domain has been replaced by synthetic DNA saturated with restriction sites. The other carries a lac P Z unit in which the natural operator has been deleted and replaced by a unique restriction site. Into this restriction site any operator can be cloned.
Our results suggest that: 1) The innermost seven base pairs of the lac operator can not be replaced without diminishing repression. 2) If Tyrl G1N2 of the recognition helix of lac repressor is substituted by Vall Ala2, the mutant repressor recognizes the mutant operator TGTAAGC GCTTACA better than the ideal or any other lac operator variant. Finally, a model is presented which may describe the binding of an alpha helix to the deep groove of B-DNA.
This is a preview of subscription content, log in via an institution.
Preview
Unable to display preview. Download preview PDF.
References
Adler, K., Beyreuther, K., Fanning, E., Geisler, N., Gronenborn, B., Klemm, A., Muller-Hill, B., Pfahl, M. and Schmitz, A., How lac repressor binds DNA, Nature 237:322 (1972).
Ohlendorf, D. H., Anderson, W.F., Fisher, R.G., Takeda, Y. and Matthews, B. W., The molecular basis of DNA-protein recognition inferred from the structure of cro repressor, Nature 298:718 (1982).
Matthews, B.W., Ohlendorf, D. H., Anderson, W.F. and Takeda, Y., Structure of the DNA-binding region of lac repressor inferred from its homology with cro repressor, Proc.Natl.Acad.Sci.USA 79:1428 (1982).
Sauer, R. T., Yocum, R. R., Doolittle, R. F., Lewis, M. and Pabo, C.O., Homology among DNA-bindig proteins suggests use of a conserved super-secondary structure, Nature 298:447 (1982).
Gicquel-Sanzey, B. and Cossart, P., Homology between different procaryotic DNA-bindig regulatory proteins and between their sites of action, The EMBO J. 1:591 (1982).
Pabo, C. O. and Lewis, M., The operator-binding domain of λ repressor: structure and DNA recognition, Nature 298:443 (1982).
McKay, D. B. and Steitz, T., Structure of catabolite activator protein at 2.9 A resolution suggests binding to left handed B-DNA, Nature 290:744 (1981).
Schevitz, R. W., Otwinowski, Z., Joachimiak, A., Lawson, D.L. and Sigler, P. B., The three-dimensional structure of trp repressor, Nature 317:782 (1985).
Youderian, P., Vershon, A., Bouvier, S., Sauer, R. T., Susskind, M., Changing the DNA-binding specificity of a repressor, Cell 35:777 (1983).
Ebright, R.H., Cossart, P., Gicquel-Sanzey, B. and Beckwith, J., Mutations that alter the DNA sequence specific specificity of the catabolite gene activator protein of E.coli, Nature 311:232 (1984).
Wharton, R. P., Brown, E. L. and Ptashne, M., Substituting an α-helix switches the sequence-specific DNA interaction of a repressor, Cell 38:361 (1984).
Wharton, R. P. and Ptashne, M., Changing the binding specificity of a repressor by redesigning an α-helix, Nature 316:601 (1985).
Muller-Hill, B., lac repressor and lac operator, Progr.Biophys.Molec.Biol. 30:227 (1975).
Miller, J. H., The lac I gene: its role in lac operon control and its use as a genetic system, in, “The Operon” ed. by. J.H. Miller and W.S. Peznikoff, cold Spring Harbor (1978).
Gilbert, W., Majors, J. and Maxam, A., How proteins recognize DNA sequences, in: Organisation and expression of chromosomes, Dahlem Konferenzen, Berlin (1976).
Simons, A., Tils, D., Wilcken-Bergmann, B.v. and Muller-Hill, B., Possible ideal lac operator: Escherichia coli 1ac operator — like sequences from eukaryotic genomes lack the central G C pair, Proc.Natl.Acad.Sci.USA 81:1624 (1984).
Sadler, J. R., Sasmor, H. and Betz, J. L., A perfectly symmetric 1ac operator binds the lac repressor very tightly, Proc.Natl.Acad.Sci.USA 80:6785 (1983).
Besse, M., Wilcken-Bergmann, B.v. and Muller-Hill, B., Synthetic lac operator mediated repression through lac repressor when introduced upstream and downstream from 1ac promoter, The EMBO J. 5:1377 (1986).
Ebright, R. H., Evidence for a contact between glutamine-18 of lac repressor and base pair 7 of lac operator, Proc.Natl.Acad.Sci.USA 83:303 (1986).
Wilcken-Bergmann, B.v. and Muller-Hill, B., Sequence of gal R gene indicates a common evolutionary origin of 1ac and gal repressor in Escherichia coli, Proc.Natl.Acad.Sci.USA 79:2427 (1982).
Irani, M. H. Orosz, L., A control element within a structural gene. The gal operon of Escherichia coli, cell 32:783 (1983).
Hochschi1d, A. and Ptashne, M., The recognition helices of λ repressor and λ cro make homologous contact with the operator, Cell 44, 925 (1986).
McClarin, J.A., Frederick, C.A., Wang, B.C, Greene, P., Boyer, H.W., Grable, J., Rosenberg, J.M., Structure of the DNA-EcoRI endonuclease recognition complex at 3A resolution, Science, in the press.
Schlotmann, M. and Beyreuther, K., Degradation of the DNA-binding domain of wild type and I − d lac repressors in Escherichia coli, Eur. J. Biochem. 95:39 (1979).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1987 Plenum Press, New York
About this chapter
Cite this chapter
Lehming, N., Sartorius, J., von Wilcken-Bergmann, B., Mueller-Hill, B. (1987). Searching for the Code of Ideal Protein-DNA-Recognition. In: Guschlbauer, W., Saenger, W. (eds) DNA—Ligand Interactions. NATO ASI Series, vol 137. Springer, Boston, MA. https://doi.org/10.1007/978-1-4684-5383-6_11
Download citation
DOI: https://doi.org/10.1007/978-1-4684-5383-6_11
Publisher Name: Springer, Boston, MA
Print ISBN: 978-1-4684-5385-0
Online ISBN: 978-1-4684-5383-6
eBook Packages: Springer Book Archive