Linkage between operator binding and dimer to octamer self-assembly of bacteriophage lambda cI repressor

被引:29
作者
Rusinova, E
Ross, JBA
Laue, TM
Sowers, LC
Senear, DF
机构
[1] UNIV CALIF IRVINE,DEPT MOL BIOL & BIOCHEM,IRVINE,CA 92697
[2] CUNY MT SINAI SCH MED,DEPT BIOCHEM,NEW YORK,NY 10029
[3] UNIV NEW HAMPSHIRE,DEPT BIOCHEM,DURHAM,NH 03824
[4] CITY HOPE NATL MED CTR,DIV PEDIAT,DUARTE,CA 91010
关键词
D O I
10.1021/bi971368k
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Cooperative binding of the bacteriophage lambda cI repressor dimer to specific sites of the phage operators O-R and O-L controls the developmental state of the phage. Cooperativity has long been thought to be mediated by self-assembly of repressor dimers to form tetramers which can bind simultaneously to adjacent operators. More recently, we demonstrated that when free repressor dimers self-associate in solution, tetramer is an intermediate in a concerted assembly reaction leading to octamer as the predominant higher order species [Senear, D. F., et al. (1993) Biochemistry 32, 6179-6189]. Even as a minority component in the assembly reaction, tetramer can account for pairwise cooperativity. In a similar manner, were it able to bind all three operators simultaneously, octamer could account for three-way cooperativity. In fact, based solely on repressor self-assembly, the naive prediction is that the repressor-O-R interactions should be substantially more cooperative than they are. Evidently, there are unfavorable contributions to cooperativity from processes other than repressor self-assembly. Here, we focus on coupling between repressor self-association and operator binding as one possible unfavorable contribution to cooperativity. Sedimentation equilibrium analysis was used to compare the dimer-octamer association reactions of a repressor dimer-O(R)1 complex and free repressor dimer. Fluorescence anisotropy was used to investigate O(R)1 binding to free dimers and dimers assembled as higher order species. The results of these experiments indicate a significant and salt-dependent unfavorable contribution generated by such coupling. Since the oligonucleotides used in these experiments are the size of single operator sites, this coupling is mediated by the protein, not by the DNA. This mechanism does not account for an additional, salt-independent, unfavorable contribution which we presume is transmitted via the DNA. Thus, unfavorable contributions generated by structural transitions in both macromolecules serve to moderate the effect of self-association alone. We speculate that this is a general feature of cooperative protein-DNA interactions.
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收藏
页码:12994 / 13003
页数:10
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共 34 条
  • [1] Badea M G, 1979, Methods Enzymol, V61, P378
  • [2] Dimer-dimer interfaces of the lambda-repressor are different in liganded and free states
    Bandyopadhyay, S
    Mukhopadhyay, C
    Roy, S
    [J]. BIOCHEMISTRY, 1996, 35 (15) : 5033 - 5040
  • [3] BANIK U, 1993, J BIOL CHEM, V268, P3938
  • [4] ELECTRON-MICROSCOPIC STUDY OF REPRESSOR OF BACTERIOPHAGE-LAMBDA AND ITS INTERACTION WITH OPERATOR-DNA
    BRACK, C
    PIRROTTA, V
    [J]. JOURNAL OF MOLECULAR BIOLOGY, 1975, 96 (01) : 139 - &
  • [5] STABILITY OF A LAC REPRESSOR MEDIATED LOOPED COMPLEX
    BRENOWITZ, M
    PICKAR, A
    JAMISON, E
    [J]. BIOCHEMISTRY, 1991, 30 (24) : 5986 - 5998
  • [6] Cantor C.R., 1980, BIOPHYSICAL CHEM 2, P838
  • [7] Cohn E. J., 1943, PROTEINS
  • [8] Creighton TE, 1993, PROTEINS STRUCTURES
  • [9] DURCHSCHLAG H, 1986, THERMODYNAMIC DATA B, P108
  • [10] Synthesis and hydrolysis of oligodeoxyribonucleotides containing 2-aminopurine
    Fujimoto, J
    Nuesca, Z
    Mazurek, M
    Sowers, LC
    [J]. NUCLEIC ACIDS RESEARCH, 1996, 24 (04) : 754 - 759