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Oligomeric properties and DNA binding specificities of repressor isoforms from the Streptomyces bacteriophage phiC31
1Department of Genetics, Queens Medical Centre, University Park, Nottingham NG7 2UH, UK.
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.
Insights
The phiC31 bacteriophage repressor gene produces three protein isoforms. These isoforms, particularly the smaller ones, interact and exhibit distinct DNA-binding specificities to conserved inverted repeats, influencing gene regulation.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- The Streptomyces temperate bacteriophage phiC31 repressor gene (gene c) expresses three protein isoforms: 74, 54, and 42 kDa.
- The 54 and 42 kDa isoforms are sufficient for conferring superinfection immunity, suggesting their critical role in phage regulation.
Purpose of the Study:
- To investigate the interaction between the 54 and 42 kDa repressor isoforms of bacteriophage phiC31.
- To determine the DNA-binding specificities of different phiC31 repressor isoforms and their complexes.
Main Methods:
- Co-purification using Ni-NTA chromatography to study protein interactions.
- In vitro analysis including chemical crosslinking and gel filtration to identify repressor complex formation (homo- and hetero-tetramers).
- DNase I footprinting assays to assess DNA-binding activity to conserved inverted repeat (CIR) elements.
Main Results:
- The 42 kDa native repressor (Nat42) and a 54 kDa histidine-tagged isoform (His54) interact in vivo.
- Homo- and hetero-tetrameric complexes of the repressor isoforms were observed in vitro.
- Nat42 showed specific binding to CIR6, while the combined Nat54&42 preparation and His54 bound to multiple CIR elements (CIR3-CIR6) with varying affinities.
Conclusions:
- The different protein isoforms expressed from the phiC31 repressor gene exhibit distinct DNA-binding specificities.
- These differential binding properties are analogous to eukaryotic transcription factors and likely contribute to the complex regulation of the phiC31 phage lifecycle.