Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1998 May 15;26(10):2457–2463. doi: 10.1093/nar/26.10.2457

Oligomeric properties and DNA binding specificities of repressor isoforms from the Streptomyces bacteriophage phiC31.

S E Wilson 1, M C Smith 1
PMCID: PMC147562  PMID: 9580700

Abstract

Three protein isoforms (74, 54 and 42 kDa) are expressed from repressor gene c in the Streptomyces temperate bacteriophage phiC31. Because expression of the two smaller isoforms, 54 and 42 kDa, is sufficient for superinfection immunity, the interaction between these isoforms was studied. The native 42 kDa repressor (Nat42) and an N-terminally 6x histidine-tagged 54 kDa isoform (His54) were shown by co-purification on a Ni-NTA column to interact in Streptomyces lividans . In vitro three repressor preparations, containing Nat42, His54 and the native 54 and 42 kDa isoforms expressed together (Nat54&42), were subjected to chemical crosslinking and gel filtration analysis. Homo- and hetero-tetramers were observed. Previous work showed that the smallest isoform bound to 17 bp operators containing aconservedinvertedrepeat (CIR) and that the CIRs were located at 16 loci throughout the phiC31 genome. One of the CIRs (CIR6) is believed to be critical for regulating the lytic pathway. The DNA binding activities of the three repressor preparations were studied using fragments containing CIRs (CIR3-CIR6) from the essential early region as templates for DNase I footprinting. Whereas Nat42 bound to CIR6, poorly to CIR5 but undetectably to CIR3 or CIR4, the Nat54&42 preparation could bind to all CIRs tested, albeit poorly to CIR3 and CIR4. The His54 isoform bound all CIRs tested. Isoforms expressed from the phiC31 repressor gene, like those which are expressed from many eukaryotic transcription factor genes, apparently have different binding specificities.

Full Text

The Full Text of this article is available as a PDF (403.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bläsi U., Nam K., Hartz D., Gold L., Young R. Dual translational initiation sites control function of the lambda S gene. EMBO J. 1989 Nov;8(11):3501–3510. doi: 10.1002/j.1460-2075.1989.tb08515.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Brown K. L., Sarkis G. J., Wadsworth C., Hatfull G. F. Transcriptional silencing by the mycobacteriophage L5 repressor. EMBO J. 1997 Oct 1;16(19):5914–5921. doi: 10.1093/emboj/16.19.5914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bruton C. J., Guthrie E. P., Chater K. F. Phage vectors that allow monitoring of transcription of secondary metabolism genes in Streptomyces. Biotechnology (N Y) 1991 Jul;9(7):652–656. doi: 10.1038/nbt0791-652. [DOI] [PubMed] [Google Scholar]
  5. Chakerian A. E., Matthews K. S. Effect of lac repressor oligomerization on regulatory outcome. Mol Microbiol. 1992 Apr;6(8):963–968. doi: 10.1111/j.1365-2958.1992.tb02162.x. [DOI] [PubMed] [Google Scholar]
  6. Chater K. F. Multilevel regulation of Streptomyces differentiation. Trends Genet. 1989 Nov;5(11):372–377. doi: 10.1016/0168-9525(89)90172-8. [DOI] [PubMed] [Google Scholar]
  7. Dandanell G., Valentin-Hansen P., Larsen J. E., Hammer K. Long-range cooperativity between gene regulatory sequences in a prokaryote. 1987 Feb 26-Mar 4Nature. 325(6107):823–826. doi: 10.1038/325823a0. [DOI] [PubMed] [Google Scholar]
  8. Epstein J. A., Glaser T., Cai J., Jepeal L., Walton D. S., Maas R. L. Two independent and interactive DNA-binding subdomains of the Pax6 paired domain are regulated by alternative splicing. Genes Dev. 1994 Sep 1;8(17):2022–2034. doi: 10.1101/gad.8.17.2022. [DOI] [PubMed] [Google Scholar]
  9. Escoubas J. M., Prère M. F., Fayet O., Salvignol I., Galas D., Zerbib D., Chandler M. Translational control of transposition activity of the bacterial insertion sequence IS1. EMBO J. 1991 Mar;10(3):705–712. doi: 10.1002/j.1460-2075.1991.tb08000.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Galas D. J., Schmitz A. DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res. 1978 Sep;5(9):3157–3170. doi: 10.1093/nar/5.9.3157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Giguère V., Tini M., Flock G., Ong E., Evans R. M., Otulakowski G. Isoform-specific amino-terminal domains dictate DNA-binding properties of ROR alpha, a novel family of orphan hormone nuclear receptors. Genes Dev. 1994 Mar 1;8(5):538–553. doi: 10.1101/gad.8.5.538. [DOI] [PubMed] [Google Scholar]
  12. Hartley N. M., Murphy G. O., Bruton C. J., Chater K. F. Sequence of the essential early region of phi C31, a temperate phage of Streptomyces spp. with unusual features in its lytic development. Gene. 1994 Sep 15;147(1):29–40. doi: 10.1016/0378-1119(94)90035-3. [DOI] [PubMed] [Google Scholar]
  13. Holmes D. J., Caso J. L., Thompson C. J. Autogenous transcriptional activation of a thiostrepton-induced gene in Streptomyces lividans. EMBO J. 1993 Aug;12(8):3183–3191. doi: 10.1002/j.1460-2075.1993.tb05987.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Howe C. W., Smith M. C. Gene expression in the cos region of the Streptomyces temperate actinophage phi C31. Microbiology. 1996 Jun;142(Pt 6):1357–1367. doi: 10.1099/13500872-142-6-1357. [DOI] [PubMed] [Google Scholar]
  15. Hsu T., Gogos J. A., Kirsh S. A., Kafatos F. C. Multiple zinc finger forms resulting from developmentally regulated alternative splicing of a transcription factor gene. Science. 1992 Sep 25;257(5078):1946–1950. doi: 10.1126/science.1411512. [DOI] [PubMed] [Google Scholar]
  16. Huo L., Martin K. J., Schleif R. Alternative DNA loops regulate the arabinose operon in Escherichia coli. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5444–5448. doi: 10.1073/pnas.85.15.5444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ingham C. J., Hunter I. S., Smith M. C. Rho-independent terminators without 3' poly-U tails from the early region of actinophage øC31. Nucleic Acids Res. 1995 Feb 11;23(3):370–376. doi: 10.1093/nar/23.3.370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ingham C. J., Owen C. E., Wilson S. E., Hunter I. S., Smith M. C. An operator associated with autoregulation of the repressor gene in actinophage phiC31 is found in highly conserved copies in intergenic regions in the phage genome. Nucleic Acids Res. 1994 Mar 11;22(5):821–827. doi: 10.1093/nar/22.5.821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kobler L., Schwertfirm G., Schmieger H., Bolotin A., Sladkova I. Construction and transduction of a shuttle vector bearing the cos site of Streptomyces phage phi C31 and determination of its cohesive ends. FEMS Microbiol Lett. 1991 Mar 1;62(2-3):347–353. doi: 10.1016/0378-1097(91)90183-b. [DOI] [PubMed] [Google Scholar]
  20. Kofoid E. C., Parkinson J. S. Tandem translation starts in the cheA locus of Escherichia coli. J Bacteriol. 1991 Mar;173(6):2116–2119. doi: 10.1128/jb.173.6.2116-2119.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kuhstoss S., Rao R. N. Analysis of the integration function of the streptomycete bacteriophage phi C31. J Mol Biol. 1991 Dec 20;222(4):897–908. doi: 10.1016/0022-2836(91)90584-s. [DOI] [PubMed] [Google Scholar]
  22. Kuhstoss S., Richardson M. A., Rao R. N. Plasmid cloning vectors that integrate site-specifically in Streptomyces spp. Gene. 1991 Jan 2;97(1):143–146. doi: 10.1016/0378-1119(91)90022-4. [DOI] [PubMed] [Google Scholar]
  23. Levin M. E., Hendrix R. W., Casjens S. R. A programmed translational frameshift is required for the synthesis of a bacteriophage lambda tail assembly protein. J Mol Biol. 1993 Nov 5;234(1):124–139. doi: 10.1006/jmbi.1993.1568. [DOI] [PubMed] [Google Scholar]
  24. Lewis M., Chang G., Horton N. C., Kercher M. A., Pace H. C., Schumacher M. A., Brennan R. G., Lu P. Crystal structure of the lactose operon repressor and its complexes with DNA and inducer. Science. 1996 Mar 1;271(5253):1247–1254. doi: 10.1126/science.271.5253.1247. [DOI] [PubMed] [Google Scholar]
  25. Lomovskaya N. D., Chater K. F., Mkrtumian N. M. Genetics and molecular biology of Streptomyces bacteriophages. Microbiol Rev. 1980 Jun;44(2):206–229. doi: 10.1128/mr.44.2.206-229.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Merryweather A., Rees C. E., Smith N. M., Wilkins B. M. Role of sog polypeptides specified by plasmid ColIb-P9 and their transfer between conjugating bacteria. EMBO J. 1986 Nov;5(11):3007–3012. doi: 10.1002/j.1460-2075.1986.tb04599.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Oehler S., Amouyal M., Kolkhof P., von Wilcken-Bergmann B., Müller-Hill B. Quality and position of the three lac operators of E. coli define efficiency of repression. EMBO J. 1994 Jul 15;13(14):3348–3355. doi: 10.1002/j.1460-2075.1994.tb06637.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sinclair R. B., Bibb M. J. The repressor gene (c) of the Streptomyces temperate phage phi c31: nucleotide sequence, analysis and functional cloning. Mol Gen Genet. 1988 Aug;213(2-3):269–277. doi: 10.1007/BF00339591. [DOI] [PubMed] [Google Scholar]
  29. Sinclair R. B., Bibb M. J. Transcriptional analysis of the repressor gene of the temperate Streptomyces phage phi C31. Gene. 1989 Dec 28;85(2):275–282. doi: 10.1016/0378-1119(89)90419-8. [DOI] [PubMed] [Google Scholar]
  30. Smith M. C., Owen C. E. Three in-frame N-terminally different proteins are produced from the repressor locus of the Streptomyces bacteriophage phi C31. Mol Microbiol. 1991 Nov;5(11):2833–2844. doi: 10.1111/j.1365-2958.1991.tb01992.x. [DOI] [PubMed] [Google Scholar]
  31. Wilson S. E., Ingham C. J., Hunter I. S., Smith M. C. Control of lytic development in the Streptomyces temperate phage phi C31. Mol Microbiol. 1995 Apr;16(1):131–143. doi: 10.1111/j.1365-2958.1995.tb02398.x. [DOI] [PubMed] [Google Scholar]
  32. York D., Ivanov V., Gan J., Filutowicz M. Translational options for the pir gene of plasmid R6K: multiple forms of the replication initiator protein pi. Gene. 1992 Jul 1;116(1):7–12. doi: 10.1016/0378-1119(92)90622-v. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES