Oligomeric properties and DNA binding specificities of repressor isoforms from the Streptomyces bacteriophage phi C31

S E Wilson, Margaret Caroline MacHin Smith

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Three protein isoforms (74, 54 and 42 kDa) are expressed from repressor gene c in the Streptomyces temperate bacteriophage oC31, 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 a conserved inverted repeat (CIR) and that the CIRs were located at 16 loci throughout the oC31 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 oC31 repressor gene, like those which are expressed from many eukaryotic transcription factor genes, apparently have different binding specificities.

Original languageEnglish
Pages (from-to)2457-2463
Number of pages7
JournalNucleic Acids Research
Volume26
Issue number10
Publication statusPublished - 15 May 1998

Keywords

  • TEMPERATE PHAGE PHI-C31
  • ESCHERICHIA-COLI
  • LYTIC DEVELOPMENT
  • EARLY REGION
  • GENE
  • PROTEIN
  • SEQUENCE
  • ACTINOPHAGE-PHI-C31
  • TRANSCRIPTION
  • CLONING

Cite this

Oligomeric properties and DNA binding specificities of repressor isoforms from the Streptomyces bacteriophage phi C31. / Wilson, S E ; Smith, Margaret Caroline MacHin.

In: Nucleic Acids Research, Vol. 26, No. 10, 15.05.1998, p. 2457-2463.

Research output: Contribution to journalArticle

Wilson, S E ; Smith, Margaret Caroline MacHin. / Oligomeric properties and DNA binding specificities of repressor isoforms from the Streptomyces bacteriophage phi C31. In: Nucleic Acids Research. 1998 ; Vol. 26, No. 10. pp. 2457-2463.
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abstract = "Three protein isoforms (74, 54 and 42 kDa) are expressed from repressor gene c in the Streptomyces temperate bacteriophage oC31, 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 a conserved inverted repeat (CIR) and that the CIRs were located at 16 loci throughout the oC31 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 oC31 repressor gene, like those which are expressed from many eukaryotic transcription factor genes, apparently have different binding specificities.",
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AB - Three protein isoforms (74, 54 and 42 kDa) are expressed from repressor gene c in the Streptomyces temperate bacteriophage oC31, 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 a conserved inverted repeat (CIR) and that the CIRs were located at 16 loci throughout the oC31 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 oC31 repressor gene, like those which are expressed from many eukaryotic transcription factor genes, apparently have different binding specificities.

KW - TEMPERATE PHAGE PHI-C31

KW - ESCHERICHIA-COLI

KW - LYTIC DEVELOPMENT

KW - EARLY REGION

KW - GENE

KW - PROTEIN

KW - SEQUENCE

KW - ACTINOPHAGE-PHI-C31

KW - TRANSCRIPTION

KW - CLONING

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EP - 2463

JO - Nucleic Acids Research

JF - Nucleic Acids Research

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