Repression of lac promoter as a function of distance, phase and quality of an auxiliary lac operator.

The tetrameric Lac repressor can bind simultaneously to two lac operators on the same DNA molecule, thereby including the formation of a DNA loop. We investigated the phasing dependence of DNA loop formation between lac operator O1 and an auxiliary ideal lac operator (O(id)) on the bacterial chromosome, with inter-operator distances varying from 57.5 to 1493.5 bp. Repression of a CAP-independent lac UV5 promoter by O1 at its natural position increased up to 50-fold in the presence of an optimally positioned auxiliary O(id)). Repression values alternated between local maxima and minima with a periodicity of 11.0 to 11.3 bp, suggesting that the chromosomal helical repeat is in this range in vivo. Repression increased significantly with decreasing inter-operator DNA length, indicating that the local Lac repressor concentration at O1 is crucial for tight repression. Maximal repression, attributed to stable DNA loop formation, was obtained at an operator spacing of 70.5 bp. Other repression maxima occurred at operator distances of 92.5 and 115.5 bp, corresponding to natural operator spacings in the lac and in the gal operon, respectively. Substitution of the auxiliary O(id) with the weaker binding lac operator O3 lowered repression efficiency, presumably due to the reduced local concentration of Lac repressor.

[1]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[2]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .

[3]  R. L. Baldwin,et al.  Energetics of DNA twisting. I. Relation between twist and cyclization probability. , 1983, Journal of molecular biology.

[4]  R. L. Baldwin,et al.  Energetics of DNA twisting. II. Topoisomer analysis. , 1983, Journal of molecular biology.

[5]  M. Simon,et al.  Host protein requirements for in vitro site-specific DNA inversion , 1986, Cell.

[6]  B. Müller-Hill,et al.  Specific destruction of the second lac operator decreases repression of the lac operon in Escherichia coli fivefold. , 1987, Journal of molecular biology.

[7]  M C Mossing,et al.  Upstream operators enhance repression of the lac promoter. , 1986, Science.

[8]  R. Gourse,et al.  A third recognition element in bacterial promoters: DNA binding by the alpha subunit of RNA polymerase. , 1993, Science.

[9]  R. Wells,et al.  Influence of supercoiling and sequence context on operator DNA binding with lac repressor. , 1987, The Journal of biological chemistry.

[10]  Record Mt,et al.  Stable DNA Loops in Vivo and in Vitro: Roles in Gene Regulation at a Distance and in Biophysical Characterization of DNA , 1990 .

[11]  J. Wang,et al.  Action at a distance along a DNA. , 1988, Science.

[12]  J. Gralla,et al.  DNA supercoiling promotes formation of a bent repression loop in lac DNA. , 1987, Journal of molecular biology.

[13]  R. Schleif.  Why should DNA loop? , 1987, Nature.

[14]  M. Haykinson,et al.  DNA looping and the helical repeat in vitro and in vivo: effect of HU protein and enhancer location on Hin invertasome assembly. , 1993, The EMBO journal.

[15]  Y. Zivanovic,et al.  Helical repeat of DNA in solution. The V curve method. , 1987, Nucleic acids research.

[16]  T. Dunn,et al.  An operator at -280 base pairs that is required for repression of araBAD operon promoter: addition of DNA helical turns between the operator and promoter cyclically hinders repression. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[17]  J. Wang,et al.  Torsional rigidity of DNA and length dependence of the free energy of DNA supercoiling. , 1984, Journal of molecular biology.

[18]  L. Liu,et al.  DNA looping alters local DNA conformation during transcription. , 1991, Journal of molecular biology.

[19]  B. Müller-Hill,et al.  lac repressor forms loops with linear DNA carrying two suitably spaced lac operators. , 1987, The EMBO journal.

[20]  M. Record,et al.  In vivo thermodynamic analysis of repression with and without looping in lac constructs. Estimates of free and local lac repressor concentrations and of physical properties of a region of supercoiled plasmid DNA in vivo. , 1993, Journal of molecular biology.

[21]  R. L. Baldwin,et al.  DNA flexibility studied by covalent closure of short fragments into circles. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[22]  J. Gralla,et al.  Probing co-operative DNA-binding in vivo. The lac O1:O3 interaction. , 1988, Journal of molecular biology.

[23]  J. Wang,et al.  Helical repeat of DNA in solution. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[24]  J. Schellman,et al.  DNA condensation with polyamines I. Spectroscopic studies. , 1978, Journal of molecular biology.

[25]  V. Bloomfield,et al.  Condensation of DNA by multivalent cations: Considerations on mechanism , 1991, Biopolymers.

[26]  B. Müller-Hill,et al.  Quality and position of the three lac operators of E. coli define efficiency of repression. , 1994, The EMBO journal.

[27]  B. Müller-Hill,et al.  The three operators of the lac operon cooperate in repression. , 1990, The EMBO journal.

[28]  N. Cozzarelli,et al.  Use of site-specific recombination as a probe of DNA structure and metabolism in vivo. , 1987, Journal of molecular biology.

[29]  D. Pettijohn,et al.  Structure and properties of the bacterial nucleoid , 1982, Cell.

[30]  B. Müller-Hill,et al.  lac repressor forms stable loops in vitro with supercoiled wild-type lac DNA containing all three natural lac operators. , 1990, Journal of molecular biology.

[31]  J. Wang,et al.  Supercoiling of the DNA template during transcription. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[32]  S. Adhya,et al.  A control element within a structural gene: The gal operon of Escherichia coli , 1983, Cell.

[33]  T. Steitz,et al.  Two helix DNA binding motif of CAP found in lac repressor and gal repressor. , 1982, Nucleic acids research.

[34]  B. Müller-Hill,et al.  Possible ideal lac operator: Escherichia coli lac operator-like sequences from eukaryotic genomes lack the central G X C pair. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[35]  T. Steitz,et al.  Crystal structure of lac repressor core tetramer and its implications for DNA looping. , 1995, Science.

[36]  A. Klug,et al.  Sequence-dependent helical periodicity of DNA , 1981, Nature.

[37]  M C Mossing,et al.  Physical properties of DNA in vivo as probed by the length dependence of the lac operator looping process. , 1988, Biochemistry.

[38]  P. Hagerman,et al.  DNA ring closure mediated by protein HU. , 1989, The Journal of biological chemistry.

[39]  B. Müller-Hill,et al.  A versatile phage lambda expression vector system for cloning in Escherichia coli. , 1989, Gene.

[40]  P. Valentin‐Hansen,et al.  Long-range cooperativity between gene regulatory sequences in a prokaryote , 1987, Nature.

[41]  H. Fritz,et al.  Characterization of two mutations in the Escherichia coli galE gene inactivating the second galactose operator and comparative studies of repressor binding. , 1983, The EMBO journal.

[42]  N. Cozzarelli,et al.  Helical repeat and linking number of surface-wrapped DNA. , 1988, Science.

[43]  K. Drlica,et al.  Histonelike proteins of bacteria. , 1987, Microbiological reviews.

[44]  R. Schleif,et al.  In vivo DNA loops in araCBAD: size limits and helical repeat. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[45]  A. Nordheim,et al.  DNA supercoiling changes the spacing requirement of two lac operators for DNA loop formation with lac repressor. , 1988, The EMBO journal.

[46]  W. Gilbert,et al.  ISOLATION OF THE LAC REPRESSOR , 1966, Proceedings of the National Academy of Sciences of the United States of America.

[47]  J. Sadler,et al.  A perfectly symmetric lac operator binds the lac repressor very tightly. , 1983, Proceedings of the National Academy of Sciences of the United States of America.