Abstract
Plasmid pLS1-encoded 45-amino acid transcriptional repressor CopG (formerly RepA) has been chemically synthesized. A one-step purification of the synthetic protein has been developed, which yields high levels of pure protein with low or no contamination of truncated products. We have compared some properties of the chemical CopG protein with those of the biologically purified CopG. The two proteins were indistinguishable in (i) their ability to generate specific protein-DNA complexes, (ii) their capacity to protect a restriction site included within the CopG DNA target, and (iii) in their in vitro capacity to specifically repress synthesis of copG mRNA.
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- Adhya S. Multipartite genetic control elements: communication by DNA loop. Annu Rev Genet. 1989;23:227–250. doi: 10.1146/annurev.ge.23.120189.001303. [DOI] [PubMed] [Google Scholar]
- Brennan R. G., Matthews B. W. The helix-turn-helix DNA binding motif. J Biol Chem. 1989 Feb 5;264(4):1903–1906. [PubMed] [Google Scholar]
- Funakoshi S., Fukuda H., Fujii N. Chemoselective one-step purification method for peptides synthesized by the solid-phase technique. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):6981–6985. doi: 10.1073/pnas.88.16.6981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kleina L. G., Miller J. H. Genetic studies of the lac repressor. XIII. Extensive amino acid replacements generated by the use of natural and synthetic nonsense suppressors. J Mol Biol. 1990 Mar 20;212(2):295–318. doi: 10.1016/0022-2836(90)90126-7. [DOI] [PubMed] [Google Scholar]
- Lacks S. A., Lopez P., Greenberg B., Espinosa M. Identification and analysis of genes for tetracycline resistance and replication functions in the broad-host-range plasmid pLS1. J Mol Biol. 1986 Dec 20;192(4):753–765. doi: 10.1016/0022-2836(86)90026-4. [DOI] [PubMed] [Google Scholar]
- Lane D., Prentki P., Chandler M. Use of gel retardation to analyze protein-nucleic acid interactions. Microbiol Rev. 1992 Dec;56(4):509–528. doi: 10.1128/mr.56.4.509-528.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lilley D. M. Transcription. When the CAP fits bent DNA. Nature. 1991 Dec 5;354(6352):359–360. doi: 10.1038/354359a0. [DOI] [PubMed] [Google Scholar]
- Merrifield B. Solid phase synthesis. Science. 1986 Apr 18;232(4748):341–347. doi: 10.1126/science.3961484. [DOI] [PubMed] [Google Scholar]
- Nash H. A. Bending and supercoiling of DNA at the attachment site of bacteriophage lambda. Trends Biochem Sci. 1990 Jun;15(6):222–227. doi: 10.1016/0968-0004(90)90034-9. [DOI] [PubMed] [Google Scholar]
- Nuez B., Rojo F., Salas M. Phage phi 29 regulatory protein p4 stabilizes the binding of the RNA polymerase to the late promoter in a process involving direct protein-protein contacts. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11401–11405. doi: 10.1073/pnas.89.23.11401. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ohlendorf D. H., Anderson W. F., Matthews B. W. Many gene-regulatory proteins appear to have a similar alpha-helical fold that binds DNA and evolved from a common precursor. J Mol Evol. 1983;19(2):109–114. doi: 10.1007/BF02300748. [DOI] [PubMed] [Google Scholar]
- Ptashne M. Gene regulation by proteins acting nearby and at a distance. Nature. 1986 Aug 21;322(6081):697–701. doi: 10.1038/322697a0. [DOI] [PubMed] [Google Scholar]
- Pérez-Martín J., del Solar G. H., Lurz R., de la Campa A. G., Dobrinski B., Espinosa M. Induced bending of plasmid pLS1 DNA by the plasmid-encoded protein RepA. J Biol Chem. 1989 Dec 15;264(35):21334–21339. [PubMed] [Google Scholar]
- Reidhaar-Olson J. F., Sauer R. T. Combinatorial cassette mutagenesis as a probe of the informational content of protein sequences. Science. 1988 Jul 1;241(4861):53–57. doi: 10.1126/science.3388019. [DOI] [PubMed] [Google Scholar]
- Rojo F., Zaballos A., Salas M. Bend induced by the phage phi 29 transcriptional activator in the viral late promoter is required for activation. J Mol Biol. 1990 Feb 20;211(4):713–725. doi: 10.1016/0022-2836(90)90072-t. [DOI] [PubMed] [Google Scholar]
- Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
- Schleif R. DNA binding by proteins. Science. 1988 Sep 2;241(4870):1182–1187. doi: 10.1126/science.2842864. [DOI] [PubMed] [Google Scholar]
- Schultz S. C., Shields G. C., Steitz T. A. Crystal structure of a CAP-DNA complex: the DNA is bent by 90 degrees. Science. 1991 Aug 30;253(5023):1001–1007. doi: 10.1126/science.1653449. [DOI] [PubMed] [Google Scholar]
- Shin J. A., Ebright R. H., Dervan P. B. Orientation of the Lac repressor DNA binding domain in complex with the left lac operator half site characterized by affinity cleaving. Nucleic Acids Res. 1991 Oct 11;19(19):5233–5236. doi: 10.1093/nar/19.19.5233. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tullius T. D., Dombroski B. A., Churchill M. E., Kam L. Hydroxyl radical footprinting: a high-resolution method for mapping protein-DNA contacts. Methods Enzymol. 1987;155:537–558. doi: 10.1016/0076-6879(87)55035-2. [DOI] [PubMed] [Google Scholar]
- del Solar G. H., Pérez-Martín J., Espinosa M. Plasmid pLS1-encoded RepA protein regulates transcription from repAB promoter by binding to a DNA sequence containing a 13-base pair symmetric element. J Biol Chem. 1990 Jul 25;265(21):12569–12575. [PubMed] [Google Scholar]
- del Solar G. H., de al Campa A. G., Pérez-Martín J., Choli T., Espinosa M. Purification and characterization of RepA, a protein involved in the copy number control of plasmid pLS1. Nucleic Acids Res. 1989 Apr 11;17(7):2405–2420. doi: 10.1093/nar/17.7.2405. [DOI] [PMC free article] [PubMed] [Google Scholar]
- del Solar G., Espinosa M. The copy number of plasmid pLS1 is regulated by two trans-acting plasmid products: the antisense RNA II and the repressor protein, RepA. Mol Microbiol. 1992 Jan;6(1):83–94. doi: 10.1111/j.1365-2958.1992.tb00840.x. [DOI] [PubMed] [Google Scholar]