Abstract
The lac and gal control regions contain two functional overlapping promoters P1 and P2. Point mutations can shift transcription from P1 to P2 and vice versa. We show that the reactivity of DNA fragments towards nucleolytic attack with orthophenanthroline cuprous complex can be used to predict which promoter competes more efficiently for RNA polymerase binding. Furthermore, similar changes in reactivity are observed as closed complexes isomerize to form the final open complexes, provided that the functional start is taken as a reference. We found a correlation between the reactivity pattern of -10 regions in uncomplexed DNA and the rate of open complex formation.
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- Aiba H., Adhya S., de Crombrugghe B. Evidence for two functional gal promoters in intact Escherichia coli cells. J Biol Chem. 1981 Nov 25;256(22):11905–11910. [PubMed] [Google Scholar]
- Amouyal M., Buc H. Topological unwinding of strong and weak promoters by RNA polymerase. A comparison between the lac wild-type and the UV5 sites of Escherichia coli. J Mol Biol. 1987 Jun 20;195(4):795–808. doi: 10.1016/0022-2836(87)90485-2. [DOI] [PubMed] [Google Scholar]
- Bingham A. H., Ponnambalam S., Chan B., Busby S. Mutations that reduce expression from the P2 promoter of the Escherichia coli galactose operon. Gene. 1986;41(1):67–74. doi: 10.1016/0378-1119(86)90268-4. [DOI] [PubMed] [Google Scholar]
- Burgess R. R., Jendrisak J. J. A procedure for the rapid, large-scall purification of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography. Biochemistry. 1975 Oct 21;14(21):4634–4638. doi: 10.1021/bi00692a011. [DOI] [PubMed] [Google Scholar]
- Busby S., Dreyfus M. Segment-specific mutagenesis of the regulatory region in the Escherichia coli galactose operon: isolation of mutations reducing the initiation of transcription and translation. Gene. 1983 Jan-Feb;21(1-2):121–131. doi: 10.1016/0378-1119(83)90154-3. [DOI] [PubMed] [Google Scholar]
- Busby S., Truelle N., Spassky A., Dreyfus M., Buc H. The selection and characterisation of two novel mutations in the overlapping promoters of the Escherichia coli galactose operon. Gene. 1984 May;28(2):201–209. doi: 10.1016/0378-1119(84)90257-9. [DOI] [PubMed] [Google Scholar]
- Conner B. N., Yoon C., Dickerson J. L., Dickerson R. E. Helix geometry and hydration in an A-DNA tetramer: IC-C-G-G. J Mol Biol. 1984 Apr 25;174(4):663–695. doi: 10.1016/0022-2836(84)90089-5. [DOI] [PubMed] [Google Scholar]
- Dickerson R. E. Base sequence and helix structure variation in B and A DNA. J Mol Biol. 1983 May 25;166(3):419–441. doi: 10.1016/s0022-2836(83)80093-x. [DOI] [PubMed] [Google Scholar]
- Dickerson R. E., Drew H. R. Structure of a B-DNA dodecamer. II. Influence of base sequence on helix structure. J Mol Biol. 1981 Jul 15;149(4):761–786. doi: 10.1016/0022-2836(81)90357-0. [DOI] [PubMed] [Google Scholar]
- Drew H. R. Structural specificities of five commonly used DNA nucleases. J Mol Biol. 1984 Jul 15;176(4):535–557. doi: 10.1016/0022-2836(84)90176-1. [DOI] [PubMed] [Google Scholar]
- Drew H. R., Travers A. A. DNA structural variations in the E. coli tyrT promoter. Cell. 1984 Jun;37(2):491–502. doi: 10.1016/0092-8674(84)90379-9. [DOI] [PubMed] [Google Scholar]
- Drew H. R., Travers A. A. Structural junctions in DNA: the influence of flanking sequence on nuclease digestion specificities. Nucleic Acids Res. 1985 Jun 25;13(12):4445–4467. doi: 10.1093/nar/13.12.4445. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Drew H. R., Weeks J. R., Travers A. A. Negative supercoiling induces spontaneous unwinding of a bacterial promoter. EMBO J. 1985 Apr;4(4):1025–1032. doi: 10.1002/j.1460-2075.1985.tb03734.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hale P., Woodward R. S., Lebowitz J. E. coli RNA polymerase promoters on superhelical SV40 DNA are highly selective targets for chemical modification. Nature. 1980 Apr 17;284(5757):640–644. doi: 10.1038/284640a0. [DOI] [PubMed] [Google Scholar]
- Hawley D. K., McClure W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. doi: 10.1093/nar/11.8.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Keilty S., Rosenberg M. Constitutive function of a positively regulated promoter reveals new sequences essential for activity. J Biol Chem. 1987 May 5;262(13):6389–6395. [PubMed] [Google Scholar]
- Kirkegaard K., Buc H., Spassky A., Wang J. C. Mapping of single-stranded regions in duplex DNA at the sequence level: single-strand-specific cytosine methylation in RNA polymerase-promoter complexes. Proc Natl Acad Sci U S A. 1983 May;80(9):2544–2548. doi: 10.1073/pnas.80.9.2544. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Klug A., Jack A., Viswamitra M. A., Kennard O., Shakked Z., Steitz T. A. A hypothesis on a specific sequence-dependent conformation of DNA and its relation to the binding of the lac-repressor protein. J Mol Biol. 1979 Jul 15;131(4):669–680. doi: 10.1016/0022-2836(79)90196-7. [DOI] [PubMed] [Google Scholar]
- Kuhnke G., Krause A., Heibach C., Gieske U., Fritz H. J., Ehring R. The upstream operator of the Escherichia coli galactose operon is sufficient for repression of transcription initiated at the cyclic AMP-stimulated promoter. EMBO J. 1986 Jan;5(1):167–173. doi: 10.1002/j.1460-2075.1986.tb04192.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kuwabara M., Yoon C., Goyne T., Thederahn T., Sigman D. S. Nuclease activity of 1,10-phenanthroline-copper ion: reaction with CGCGAATTCGCG and its complexes with netropsin and EcoRI. Biochemistry. 1986 Nov 18;25(23):7401–7408. doi: 10.1021/bi00371a023. [DOI] [PubMed] [Google Scholar]
- Lefevre J. F., Lane A. N., Jardetzky O. Nuclear magnetic resonance study of the proton exchange rate in the operator-promoter DNA sequence of the trp operon of Escherichia coli. J Mol Biol. 1985 Oct 20;185(4):689–699. doi: 10.1016/0022-2836(85)90054-3. [DOI] [PubMed] [Google Scholar]
- Lomonossoff G. P., Butler P. J., Klug A. Sequence-dependent variation in the conformation of DNA. J Mol Biol. 1981 Jul 15;149(4):745–760. doi: 10.1016/0022-2836(81)90356-9. [DOI] [PubMed] [Google Scholar]
- Lowe P. A., Hager D. A., Burgess R. R. Purification and properties of the sigma subunit of Escherichia coli DNA-dependent RNA polymerase. Biochemistry. 1979 Apr 3;18(7):1344–1352. doi: 10.1021/bi00574a034. [DOI] [PubMed] [Google Scholar]
- McClure W. R. Mechanism and control of transcription initiation in prokaryotes. Annu Rev Biochem. 1985;54:171–204. doi: 10.1146/annurev.bi.54.070185.001131. [DOI] [PubMed] [Google Scholar]
- Patel D. J., Kozlowski S. A., Marky L. A., Broka C., Rice J. A., Itakura K., Breslauer K. J. Premelting and melting transitions in the d(CGCGAATTCGCG) self-complementary duplex in solution. Biochemistry. 1982 Feb 2;21(3):428–436. doi: 10.1021/bi00532a002. [DOI] [PubMed] [Google Scholar]
- Ponnambalam S., Spassky A., Busby S. Studies with the Escherichia coli galactose operon regulatory region carrying a point mutation that simultaneously inactivates the two overlapping promoters. Interactions with RNA polymerase and the cyclic AMP receptor protein. FEBS Lett. 1987 Jul 13;219(1):189–196. doi: 10.1016/0014-5793(87)81214-0. [DOI] [PubMed] [Google Scholar]
- Pope L. E., Sigman D. S. Secondary structure specificity of the nuclease activity of the 1,10-phenanthroline-copper complex. Proc Natl Acad Sci U S A. 1984 Jan;81(1):3–7. doi: 10.1073/pnas.81.1.3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
- Schaeffer F., Kolb A., Buc H. Point mutations change the thermal denaturation profile of a short DNA fragment containing the lactose control elements. Comparison between experiment and theory. EMBO J. 1982;1(1):99–105. doi: 10.1002/j.1460-2075.1982.tb01131.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Siebenlist U. RNA polymerase unwinds an 11-base pair segment of a phage T7 promoter. Nature. 1979 Jun 14;279(5714):651–652. doi: 10.1038/279651a0. [DOI] [PubMed] [Google Scholar]
- Siebenlist U., Simpson R. B., Gilbert W. E. coli RNA polymerase interacts homologously with two different promoters. Cell. 1980 Jun;20(2):269–281. doi: 10.1016/0092-8674(80)90613-3. [DOI] [PubMed] [Google Scholar]
- Sigman D. S., Graham D. R., D'Aurora V., Stern A. M. Oxygen-dependent cleavage of DNA by the 1,10-phenanthroline . cuprous complex. Inhibition of Escherichia coli DNA polymerase I. J Biol Chem. 1979 Dec 25;254(24):12269–12272. [PubMed] [Google Scholar]
- Sigman D. S., Spassky A., Rimsky S., Buc H. Conformational analysis of lac promoters using the nuclease activity of 1,10-phenanthroline-copper ion. Biopolymers. 1985 Jan;24(1):183–197. doi: 10.1002/bip.360240115. [DOI] [PubMed] [Google Scholar]
- Spassky A., Busby S., Buc H. On the action of the cyclic AMP-cyclic AMP receptor protein complex at the Escherichia coli lactose and galactose promoter regions. EMBO J. 1984 Jan;3(1):43–50. doi: 10.1002/j.1460-2075.1984.tb01759.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Spassky A., Sigman D. S. Nuclease activity of 1,10-phenanthroline-copper ion. Conformational analysis and footprinting of the lac operon. Biochemistry. 1985 Dec 31;24(27):8050–8056. doi: 10.1021/bi00348a032. [DOI] [PubMed] [Google Scholar]
- Stefano J. E., Gralla J. D. Spacer mutations in the lac ps promoter. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1069–1072. doi: 10.1073/pnas.79.4.1069. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Travers A. A. Structure and function of E. coli promoter DNA. CRC Crit Rev Biochem. 1987;22(3):181–219. doi: 10.3109/10409238709101483. [DOI] [PubMed] [Google Scholar]
- Tullius T. D., Dombroski B. A. Hydroxyl radical "footprinting": high-resolution information about DNA-protein contacts and application to lambda repressor and Cro protein. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5469–5473. doi: 10.1073/pnas.83.15.5469. [DOI] [PMC free article] [PubMed] [Google Scholar]