Changes in the DNA structure of the lac UV5 promoter during formation of an open complex with Escherichia coli RNA polymerase.

By chemical and enzymatic methods, two stable complexes between Escherichia coli RNA polymerase and a linear DNA fragment carrying the lac UV5 promoter have been identified. In these binary complexes, DNA can adopt two alternate conformations as a function of temperature. Contacts between RNA polymerase and the DNA phosphate backbone are indistinguishable in these two forms, as revealed by probing with pancreatic DNase I. Protection of enhancement of the reactivity of the bases toward (CH3)2SO4 occurs, however, only in the form that predominates above 22 degrees C, RPo. The form stable at low temperature, RPi, is a "closed" complex since no single-stranded region is detectable in the DNA. The strong temperature dependence of the equilibrium constant, the midpoint value of the transition, and the rate of conversion between these two forms are in close agreement with a series of measurements performed by using a transcriptional assay and reported in the preceding paper [Buc, H., & McClure, W. R. (1985) Biochemistry (preceding paper in this issue)]. These data further support the postulated mechanism of open complex formation involving three sequential steps: R + P in equilibrium RPc in equilibrium RPi in equilibrium RPo. The binary complex RPc, which accumulates transiently at 37 degrees C before the isomerization leading to open complex formation, is not significantly protected against enzymatic cleavage or chemical modification and is therefore distinct from RPi and RPo.(ABSTRACT TRUNCATED AT 250 WORDS)

[1]  Howard B. Gamper,et al.  A topological model for transcription based on unwinding angle analysis of E. coli RNA polymerase binary, initiation and ternary complexes , 1982, Cell.

[2]  F. Young.  Biochemistry , 1955, The Indian Medical Gazette.

[3]  W. McClure,et al.  Mechanism of CRP-cAMP activation of lac operon transcription initiation activation of the P1 promoter. , 1984, Journal of molecular biology.

[4]  A. Mirzabekov,et al.  Topography of interaction of Escherichia coli RNA polymerase subunits with lac UV5 promoter , 1981, FEBS letters.

[5]  J. Wang,et al.  Mapping of single-stranded regions in duplex DNA at the sequence level: single-strand-specific cytosine methylation in RNA polymerase-promoter complexes. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[6]  L. Johnsrud.  Contacts between Escherichia coli RNA polymerase and a lac operon promoter. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[7]  R. Burgess,et al.  A procedure for the rapid, large-scall purification of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography. , 1975, Biochemistry.

[8]  H. Bujard,et al.  Interaction of E. coli RNA polymerase with promotors of coliphage T5 , 1977, Molecular and General Genetics MGG.

[9]  B. Sanzey,et al.  Modulation of gene expression by drugs affecting deoxyribonucleic acid gyrase , 1979, Journal of bacteriology.

[10]  W. Gilbert,et al.  Contacts between Escherichia coli RNA polymerase and an early promoter of phage T7. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

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

[12]  P. Seeburg,et al.  Interaction of RNA polymerase with promoters from bacteriophage fd. , 1977, European journal of biochemistry.

[13]  J. Gralla,et al.  Spacer mutations in the lac ps promoter. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[14]  D. Galas,et al.  DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. , 1978, Nucleic acids research.

[15]  R. Burgess,et al.  Purification and properties of the sigma subunit of Escherichia coli DNA-dependent RNA polymerase. , 1979, Biochemistry.

[16]  Walter Gilbert,et al.  E. coli RNA polymerase interacts homologously with two different promoters , 1980, Cell.

[17]  Elmer S. West.  From the U. S. A. , 1965 .

[18]  David J. Galas,et al.  The interaction of RNA polymerase and lac repressor with the lac control region , 1979, Nucleic Acids Res..

[19]  James C. Wang,et al.  Use of light for footprinting DNA in vivo , 1984, Nature.

[20]  H. Bujard,et al.  Interaction of Escherichia coli RNA polymerase with promoters of several coliphage and plasmid DNAs. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[21]  R. Simpson,et al.  The molecular topography of rna polymerase-promoter interaction , 1979, Cell.