An alkaline phosphatase protection assay to investigate trp repressor/operator interactions.

We have used an alkaline phosphatase protection assay to investigate the interaction of the trp repressor with its operator sequence. The assay is based on the principle that the trp repressor will protect a terminally 5'-32P-labeled operator DNA fragment from attack by alkaline phosphatase. The optimal oligonucleotide for investigating the trp repressor/operator interaction extends two base pairs from each end of the genetically defined target sequence predicted by in vivo studies [Bass et al. (1987) Genes Dev. 1, 565-572]. The assay works well over a 10,000-fold range of protein/DNA affinity and is used to show that the corepressor, L-tryptophan, causes the liganded repressor to bind a 20 base pair trp operator duplex 6400 times more strongly than the unliganded aporepressor. The affinity of the trp repressor for operators containing symmetrical mutations was interpreted in terms of the trp repressor/operator crystal structure as follows: (1) Direct hydrogen bonds with the functional groups of G-9 of the trp operator and the side chain of Arg 69 of the trp repressor contribute to DNA-binding specificity. (2) G-6 of the trp operator is critical for DNA-binding specificity probably because of the two water-mediated hydrogen bonds between its functional groups and the N-terminus of the trp repressor's E-helix. (3) Sequence-dependent aspects of the trp operator's conformation help stabilize the trp repressor/operator complex.(ABSTRACT TRUNCATED AT 250 WORDS)

[1]  P. Sigler,et al.  Stereochemical effects of L-tryptophan and its analogues on trp repressor's affinity for operator-DNA. , 1989, The Journal of biological chemistry.

[2]  O. Jardetzky,et al.  NMR assignments for the amino-terminal residues of trp repressor and their role in DNA binding. , 1989, Biochemistry.

[3]  G. A. Thomas,et al.  Environmentally induced conformational changes in B-type DNA: comparison of the conformation of the oligonucleotide d(TCGCGAATTCGCG) in solution and in its crystalline complex with the restriction nuclease EcoRI. , 1989, Biochemistry.

[4]  J. Carey.  trp repressor arms contribute binding energy without occupying unique locations on DNA. , 1989, The Journal of biological chemistry.

[5]  M Ptashne,et al.  Recognition of a DNA operator by the repressor of phage 434: a view at high resolution , 1988, Science.

[6]  Jordan,et al.  Structure of the lambda complex at 2.5 A resolution: details of the repressor-operator interactions , 1988, Science.

[7]  S. Harrison,et al.  Structure of a phage 434 Cro/DNA complex , 1988, Nature.

[8]  P. Youderian,et al.  Mutant Trp repressors with new DNA-binding specificities. , 1988, Science.

[9]  A. Joachimiak,et al.  Crystal structure of trp represser/operator complex at atomic resolution , 1988, Nature.

[10]  J. Carey,et al.  Gel retardation at low pH resolves trp repressor-DNA complexes for quantitative study. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[11]  P. Youderian,et al.  DNA specificity determinants of Escherichia coli tryptophan repressor binding. , 1987, Genes & development.

[12]  L. S. Klig,et al.  Analysis of trp repressor-operator interaction by filter binding , 1987, Nucleic Acids Res..

[13]  A. Joachimiak,et al.  The crystal structure of trp aporepressor at 1.8 Å shows how binding tryptophan enhances DNA affinity , 1987, Nature.

[14]  A. Joachimiak,et al.  Crystals of the trp repressor-operator complex suitable for X-ray diffraction analysis. , 1987, The Journal of biological chemistry.

[15]  P. Youderian,et al.  General selection for specific DNA-binding activities. , 1986, Genetics.

[16]  B. Matthews,et al.  Crystallization of a complex of cro repressor with a 17 base-pair operator. , 1986, Journal of molecular biology.

[17]  Hamilton O. Smith,et al.  Preliminary x‐ray diffraction analysis of HhaII endonuclease‐DNA cocrystals , 1986, Proteins.

[18]  J. Rosenberg,et al.  Kinked DNA in crystalline complex with EcoRI endonuclease , 1984, Nature.

[19]  H. Khorana,et al.  Specific amino acid substitutions in bacterioopsin: Replacement of a restriction fragment in the structural gene by synthetic DNA fragments containing altered codons. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[20]  C. Yanofsky,et al.  Purification and characterization of trp aporepressor. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[21]  R. Sauer,et al.  The N-terminal arms of λ repressor wrap around the operator DNA , 1982, Nature.

[22]  D. Crothers,et al.  Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. , 1981, Nucleic acids research.

[23]  M. M. Garner,et al.  A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system , 1981, Nucleic Acids Res..

[24]  W. Gilbert,et al.  A new method for sequencing DNA. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[25]  A. Riggs,et al.  Lac repressor-operator interaction. I. Equilibrium studies. , 1970, Journal of molecular biology.

[26]  S. Harrison,et al.  Structure of the represser–operator complex of bacteriophage 434 , 1987, Nature.

[27]  Catherine L. Lawson,et al.  The three-dimensional structure of trp repressor , 1985, Nature.

[28]  T Nakagawa,et al.  A pharmacokinetic analysis program (multi) for microcomputer. , 1981, Journal of pharmacobio-dynamics.

[29]  J. Van De Sande,et al.  5'-32P labeling of RNA and DNA restriction fragments. , 1980, Methods in enzymology.

[30]  W. Gilbert,et al.  Sequencing end-labeled DNA with base-specific chemical cleavages. , 1980, Methods in enzymology.

[31]  John A. Nelder,et al.  A Simplex Method for Function Minimization , 1965, Comput. J..