Abstract
The binding stoichiometries of the complexes formed when the E. coli cyclic AMP receptor protein (CAP) binds to 203 bp lac promoter-operator restriction fragments have been determined. Under quantitative binding conditions, a single dimer of CAP occupies each of two sites in the promoter. Different electrophoretic mobilities are observed for 1:1 complexes formed with L8-UV5 mutant, L305 mutant, and wild type promoter fragments, indicating sequence-specific structural differences between the complexes. The differences in gel mobility between L8-UV5 and wild type complexes disappear when the promoter fragments are cleaved with Hpa II restriction endonuclease. Models in which CAP alters DNA conformation or in which CAP forms a transient intramolecular bridge between two domains of a DNA molecule could account for these observations. The selective binding of RNA polymerase to CAP-promoter complexes is demonstrated: the binding of a single CAP dimer to the promoter is sufficient to stimulate subsequent polymerase binding. Functional CAP molecules are not released from the promoter on polymerase binding.
Full text
PDF![141](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/058c/325695/9f244aed03df/nar00346-0147.png)
![142](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/058c/325695/7b9f61ce0572/nar00346-0148.png)
![143](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/058c/325695/c0c148b7dab2/nar00346-0149.png)
![144](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/058c/325695/b0ca041c9c0a/nar00346-0150.png)
![145](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/058c/325695/62ec7f4b8f44/nar00346-0151.png)
![146](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/058c/325695/6ba7d24beeaf/nar00346-0152.png)
![147](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/058c/325695/c4c8391c279e/nar00346-0153.png)
![148](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/058c/325695/d385155f1b43/nar00346-0154.png)
![149](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/058c/325695/0da856c06834/nar00346-0155.png)
![150](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/058c/325695/90cdb1a80f68/nar00346-0156.png)
![151](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/058c/325695/286c0b64330a/nar00346-0157.png)
![152](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/058c/325695/63ee593bf401/nar00346-0158.png)
![153](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/058c/325695/d5505ecd324a/nar00346-0159.png)
![154](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/058c/325695/aab191b9db2e/nar00346-0160.png)
![155](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/058c/325695/e1f6335a78da/nar00346-0161.png)
![156](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/058c/325695/cafc1f46d6e8/nar00346-0162.png)
![157](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/058c/325695/dd005923190a/nar00346-0163.png)
![158](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/058c/325695/7ce59e3c5f5d/nar00346-0164.png)
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bresloff J. L., Crothers D. M. DNA-ethidium reaction kinetics: demonstration of direct ligand transfer between DNA binding sites. J Mol Biol. 1975 Jun 15;95(1):103–123. doi: 10.1016/0022-2836(75)90339-3. [DOI] [PubMed] [Google Scholar]
- Clewell D. B. Nature of Col E 1 plasmid replication in Escherichia coli in the presence of the chloramphenicol. J Bacteriol. 1972 May;110(2):667–676. doi: 10.1128/jb.110.2.667-676.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Culard F., Maurizot J. C. Lac repressor - lac operator interaction. Circular dichroism study. Nucleic Acids Res. 1981 Oct 10;9(19):5175–5184. doi: 10.1093/nar/9.19.5175. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dickson R. C., Abelson J., Barnes W. M., Reznikoff W. S. Genetic regulation: the Lac control region. Science. 1975 Jan 10;187(4171):27–35. doi: 10.1126/science.1088926. [DOI] [PubMed] [Google Scholar]
- Eisinger J., Blumberg W. E. Binding constants from zone transport of interacting molecules. Biochemistry. 1973 Sep 11;12(19):3648–3662. doi: 10.1021/bi00743a013. [DOI] [PubMed] [Google Scholar]
- Emmer M., deCrombrugghe B., Pastan I., Perlman R. Cyclic AMP receptor protein of E. coli: its role in the synthesis of inducible enzymes. Proc Natl Acad Sci U S A. 1970 Jun;66(2):480–487. doi: 10.1073/pnas.66.2.480. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Garner M. M., Revzin A. A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 1981 Jul 10;9(13):3047–3060. doi: 10.1093/nar/9.13.3047. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hogan M., Dattagupta N., Crothers D. M. Transmission of allosteric effects in DNA. Nature. 1979 Apr 5;278(5704):521–524. doi: 10.1038/278521a0. [DOI] [PubMed] [Google Scholar]
- Hopkins J. D. A new class of promoter mutations in the lactose operon of Escherichia coli. J Mol Biol. 1974 Aug 25;87(4):715–724. doi: 10.1016/0022-2836(74)90080-1. [DOI] [PubMed] [Google Scholar]
- Ippen K., Miller J. H., Scaife J., Beckwith J. New controlling element in the Lac operon of E. coli. Nature. 1968 Mar 2;217(5131):825–827. doi: 10.1038/217825a0. [DOI] [PubMed] [Google Scholar]
- Le Grice S. F., Matzura H. Binding of RNA polymerase and the catabolite gene activator protein within the cat promoter in Escherichia coli. J Mol Biol. 1981 Aug 5;150(2):185–196. doi: 10.1016/0022-2836(81)90448-4. [DOI] [PubMed] [Google Scholar]
- Majerfeld I. H., Miller D., Spitz E., Rickenberg H. V. Regulation of the synthesis of adenylate cyclase in Escherichia coli by the cAMP -- cAMP receptor protein complex. Mol Gen Genet. 1981;181(4):470–475. doi: 10.1007/BF00428738. [DOI] [PubMed] [Google Scholar]
- Majors J. Specific binding of CAP factor to lac promoter DNA. Nature. 1975 Aug 21;256(5519):672–674. doi: 10.1038/256672a0. [DOI] [PubMed] [Google Scholar]
- Mallick U., Herrlich P. Regulation of synthesis of a major outer membrane protein: cyclic AMP represses Escherichia coli protein III synthesis. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5520–5523. doi: 10.1073/pnas.76.11.5520. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
- McKay D. B., Fried M. G. Crystallization and preliminary X-ray diffraction data for the cyclic AMP receptor protein of Escherichia coli. J Mol Biol. 1980 May 5;139(1):95–96. doi: 10.1016/0022-2836(80)90118-7. [DOI] [PubMed] [Google Scholar]
- McKay D. B., Steitz T. A. Structure of catabolite gene activator protein at 2.9 A resolution suggests binding to left-handed B-DNA. Nature. 1981 Apr 30;290(5809):744–749. doi: 10.1038/290744a0. [DOI] [PubMed] [Google Scholar]
- O'Gorman R. B., Dunaway M., Matthews K. S. DNA binding characteristics of lactose repressor and the trypsin-resistant core repressor. J Biol Chem. 1980 Nov 10;255(21):10100–10106. [PubMed] [Google Scholar]
- O'Neill M. C., Amass K., de Crombrugghe B. Molecuar model of the DNA interaction site for the cyclic AMP receptor protein. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2213–2217. doi: 10.1073/pnas.78.4.2213. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ogden S., Haggerty D., Stoner C. M., Kolodrubetz D., Schleif R. The Escherichia coli L-arabinose operon: binding sites of the regulatory proteins and a mechanism of positive and negative regulation. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3346–3350. doi: 10.1073/pnas.77.6.3346. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Penefsky H. S. Reversible binding of Pi by beef heart mitochondrial adenosine triphosphatase. J Biol Chem. 1977 May 10;252(9):2891–2899. [PubMed] [Google Scholar]
- Rice R. H., Means G. E. Radioactive labeling of proteins in vitro. J Biol Chem. 1971 Feb 10;246(3):831–832. [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]
- Saxe S. A., Revzin A. Cooperative binding to DNA of catabolite activator protein of Escherichia coli. Biochemistry. 1979 Jan 23;18(2):255–263. doi: 10.1021/bi00569a003. [DOI] [PubMed] [Google Scholar]
- Schmitz A. Cyclic AMP receptor proteins interacts with lactose operator DNA. Nucleic Acids Res. 1981 Jan 24;9(2):277–292. doi: 10.1093/nar/9.2.277. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schmitz A., Galas D. J. The interaction of RNA polymerase and lac repressor with the lac control region. Nucleic Acids Res. 1979 Jan;6(1):111–137. doi: 10.1093/nar/6.1.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sedmak J. J., Grossberg S. E. A rapid, sensitive, and versatile assay for protein using Coomassie brilliant blue G250. Anal Biochem. 1977 May 1;79(1-2):544–552. doi: 10.1016/0003-2697(77)90428-6. [DOI] [PubMed] [Google Scholar]
- Shore D., Langowski J., Baldwin R. L. DNA flexibility studied by covalent closure of short fragments into circles. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4833–4837. doi: 10.1073/pnas.78.8.4833. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Silverstone A. E., Arditti R. R., Magasanik B. Catabolite-insensitive revertants of lac promoter mutants. Proc Natl Acad Sci U S A. 1970 Jul;66(3):773–779. doi: 10.1073/pnas.66.3.773. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Simpson R. B. Interaction of the cAMP receptor protein with the lac promoter. Nucleic Acids Res. 1980 Feb 25;8(4):759–766. [PMC free article] [PubMed] [Google Scholar]
- Sobell H. M., Tsai C. C., Gilbert S. G., Jain S. C., Sakore T. D. Organization of DNA in chromatin. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3068–3072. doi: 10.1073/pnas.73.9.3068. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Takahashi M., Blazy B., Baudras A. Non-specific interactions of CRP from E. coli with native and denatured DNAs: control of binding by cAMP and cGMP and by cation concentration. Nucleic Acids Res. 1979 Nov 24;7(6):1699–1712. doi: 10.1093/nar/7.6.1699. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Taniguchi T., O'Neill M., de Crombrugghe B. Interaction site of Escherichia coli cyclic AMP receptor protein on DNA of galactose operon promoters. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5090–5094. doi: 10.1073/pnas.76.10.5090. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wu H. M., Dattagupta N., Crothers D. M. Solution structural studies of the A and Z forms of DNA. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6808–6811. doi: 10.1073/pnas.78.11.6808. [DOI] [PMC free article] [PubMed] [Google Scholar]