Summary
Coliphage 434 tof protein was purified to a substantially pure state from λimm 434 cI dv carrier cells. The minimum molecular weight is 7,500±500 as estimated by polyacrylamide gel electrophoresis. The amino acid sequence of the nine NH2-terminal residues was determined, by manual Edman degradation of the intact protein, to be Met-Gln-Thr-Leu-Ser-Glu-Arg-Leu-(Lys)-.
The purified protein at low concentrations binds specifically to λimm 434dv DNA and at high concentrations also binds to λimm 21dv and λdv DNA. The curve of the specific binding is of Michaelis type, while that of the nonspecific binding is sigmoidal. The specific binding does not show marked temperature dependency at 4°–37°C. We have analyzed the equilibrium and kinetic data of specific binding. The equilibrium dissociation constant is 1.9x10-11M at 0°C. The association rate constant and the dissociation rate constant are 1.1–2.9x108M-1s-1 and 2.7x10-3s-1, respectively, at 0°C. The half life of the tof protein-operator DNA complex is 260s. These results suggest that the tof protein-operator interaction is much weaker than the interaction between the cI repressor and the operator reported by other workers.
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References
Anderson WF, Ohlendorf DH, Takeda Y, Matthews BW (1981) Structure of the cro repressor from bacteriophage λ and its interaction with DNA. Nature 290:754–758
Aono J, Horiuchi T (1977) Purification and some properties of presumptive tof gene product of coli phage 434. Mol Gen Genet 156:195–201
Aono J, Iwanaga S, Horiuchi T (in preparation) New 434-specific DNA binding protein copurified with the 434 tof protein from λimm 434cI dv carrier cells of Escherichia coli
Ballivet M, Reichardt LF, Eisen H (1977) Purification and properties of coliphage 21 repressor. Eur J Biochem 73:601–606
Chadwick P, Pirrotta V, Steinberg R, Hopkins N, Ptashne M (1970) The λ and 434 phage repressors. Cold Spring Harbour Symp Quant Biol 35:283–294
Chou PY, Fasman GD (1978) Prediction of the secondary structure of proteins from their amino acid sequence. In: Meister A (ed) Advances in enzymology, vol 47. Wiley & Sons, New York, pp 45–148
Cukier-Kahn R, Jacquet M, Gros F (1972) Two heat-resistant, low molecular weight proteins from Escherichia coli that stimulate DNA-directed RNA synthesis. Proc Natl Acad Sci USA 69:3643–3647
Edman P (1970) Sequence determination. In: Needleman SB (ed) Protein sequence determination. Springer, Berlin Heidelberg New York, pp 211–255
Gray WR (1972) Sequence analysis with dansyl chloride. In: Hirs CHW, Timasheff SN (eds) Methods in enzymology, vol XXV. Academic Press, New York London, pp 333–344
Hippel PH von, Revzin A, Gross CA, Wang AC (1974) Nonspecific DNA binding of genome regulating proteins as a biological control mechanisms: I. The lac operon: equilibrium aspects. Proc Natl Acad Sci USA 71:4808–4812
Hsiang MW, Cole RD, Takeda Y, Echols H (1977) Amino acid sequence of Cro regulatory protein of bacteriophage lambda. Nature 270:275–277
Iwai K, Hayashi H, Ishikawa K (1972) Calf thymus lysine- and serine-rich histone III. Complete amino acid sequence and its implication for interactions of histones with DNA. J Biochem 72:357–367
Jeppsson JO, Sjöquist J (1967) Thin-layer chromatography of PTH amino acids. Anal Biochem 18:264–269
Johnson GG, Geiduschek EP (1972) Purification of the bacteriophage SPO1 transcription factor 1. J Biol Chem 247:3571–3578
Kaiser AD, Jacob F (1957) Recombination between related temperate bacteriophages and the genetic control of immunity and prophage localization. Virology 4:509–521
Kimura S (1974) Amino acid sequence analysis for small amount of peptide by dansyl-Edman method. Japan Analyst (in Japanese) 23:563–575
Lin SY, Riggs AD (1975) The general affinity of lac repressor for E. coli DNA: Implications for gene regulation in prokaryotes and eukaryotes. Cell 4:107–112
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Matsubara K, Takagi Y, Mukai T (1975) In vitro construction of different oligomeric forms of λdv DNA and studies on their transforming activities. J Virol 16:479–485
Matsubara K, Otsuji Y (1978) Preparation of plasmids from lambdoid phages and studies on their incompatibilities. Plasmid 1:284–296
Murotsu T, Tanaka H, Imaji M, Koga H, Matsubara K, Horiuchi T (1977) Purification and properties of a λ operator-binding protein which is expected to be autorepressor (tof protein) from E. coli carrying λdv plasmid. Mol Gen Genet 157:139–147
Ovchinnikov YA, Guryev SO, Krayev AS, Monastyrskaya GS, Skryabin KG, Sverdlov ED, Zakharyev VM, Bayev AA (1979) Primary structure of an Eco R1 fragment of λimm 434 DNA containing regions cI-cro of phage 434 and cII-O of phage lambda. Gene 6:235–249
Pirrotta V, Ptashne M (1969) Isolation of the 434 phage repressor. Nature 222:541–544
Pirrotta V (1979) Operators and promoters in the O R region of phage 434. Nucl Acids Res 6:1495–1508
Reisfeld RA, Lewis UJ, Williams DE (1962) Disk electrophoresis of basic proteins and peptides on polyacrylamide gels. Nature 195:281–283
Riggs AD, Bourgeois S, Newby RF, Cohn M (1968) DNA binding of the lac repressor. J Mol Biol 34:365–368
Riggs AD, Suzuki H, Bourgeois S (1970a) lac repressor-operator interaction 1. Equilibrium studies. J Mol Biol 48:67–83
Riggs AD, Bourgeois S, Cohn M (1970b) The lac repressor-operator interaction III. Kinetic studies. J Mol Biol 53:401–417
Rouvière-Yaniv J, Gros F (1975) Characterization of a novel, lowmolecular-weight DNA-binding protein from Escherichia coli. Proc Natl Acad Sci USA 72:3428–3432
Searcy DG (1975) Histone-like protein in the prokaryote Thermoplasma acidophilum. Biochim Biophys Acta 395:535–547
Simon MN, Davis RW, Davidson N (1971) Heteroduplexes of DNA molecules of lambdoid phages: physical mapping of their base sequence relationship by electron microscopy. In: Hershey AD (ed) The bacteriophage Lambda. Cold Spring Harbor Laboratory, New York, pp 313–328
Smith MH (1968) Table 1. Full data-globular proteins, Table III Incomplete data-globular proteins. In: Sober HA (ed) Handbook of biochemistry. The Chemical Rubber Co., Cleaveland, Ohio, pp C-10–23
Spackman DH, Stein WH, Moore S (1958) Automatic recording apparatus for use in the chromatography of amino acids. Anal Chem 30:1190–1206
Takeda Y, Folkmanis A, Echols H (1977) The cro regulatory protein specified by bacteriophage λ. Structure DNA-binding, and repression of RNA synthesis. J Biol Chem 252:6177–6183
Weber K, Pringle JR, Osborn M (1972) Measurement of molecular weights by electrophoresis on SDS-acrylamide gel. In: Hirs CHW, Timasheff SN (eds) Methods in enzymology, vol XXVI. Academic Press, New York, pp 3–27
Wilcox G, Clemetson KJ, Cleary P, Englesberg E (1974) Interaction of the regulatory gene product with the operator site in the L-arabinose operon of Escherichia coli. J Mol Biol 85:589–602
Woods KR, Wang KT (1967) Separation of dansyl-amino acids by polyamide layer chromatography. Biochim Biophys Acta 133:367–370
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Aono, J., Kangawa, K., Matsuo, H. et al. Coliphage 434 tofprotein: NH2-terminal amino acid sequence and kinetic and equilibrium measurements of DNA binding. Mol Gen Genet 186, 460–466 (1982). https://doi.org/10.1007/BF00337948
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DOI: https://doi.org/10.1007/BF00337948