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- Ahlgren-Berg, Alexandra, et al.
(författare)
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A comparison of the DNA binding and bending capacities and the oligomeric states of the immunity repressors of heteroimmune coliphages P2 and W Phi
- 2007
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 35:10, s. 3167-3180
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Tidskriftsartikel (refereegranskat)abstract
- Bacteriophages P2 and W Phi are heteroimmune members of the P2-like family of temperate Escherichia coli phages. Temperate phages can grow lytically or form lysogeny after infection. A transcriptional switch that contains two convergent promoters, Pe and Pc, and two repressors regulate what life mode to enter. The immunity repressor C is the first gene of the lysogenic operon, and it blocks the early Pe promoter. In this work, some characteristics of the C proteins of P2 and W Phi are compared. An in vivo genetic analysis shows that W Phi C, like P2C, has a strong dimerization activity in the absence of its DNA target. Both C proteins recognize two directly repeated sequences, termed half-sites and a strong bending is induced in the respective DNA target upon binding. P2C is unable to bind to one half-site as opposed to W Phi, but both half-sites are required for repression of W Phi Pe. A reduction from three to two helical turns between the centers of the half-sites in W Phi has no significant effect on the capacity to repress Pe. However, the protein-DNA complexes formed differ, as determined by electrophoretic mobility shift experiments. A difference in spontaneous phage production is observed in isogenic lysogens.
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| 6. |
- Henriksson Peltola, Petri, 1965-
(författare)
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Molecular Studies of Three Coliphage Repressor Proteins P2 C, P2 Hy dis C and Wphi C : Kinetics, Oligomeric States and Structural Studies
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- P2, P2 Hy dis and WΦ are heteroimmune and closely related E. coli phages that can either grow lytically or form lysogeny after infection. The developmental switch region that contains two divergent promoters, the early Pe and the lysogenic Pc promoter, and two repressors, controls the life mode of the phage. The first gene product from the lysogenic operon is the immunity C repressor protein which recognizes an operator consisting of two direct repeats (denoted half sites) flanking the -35 and -10 regions of Pe. Though the identity of the structural genes is > 96%, the immunity C repressors only show the between 43-63% identity. P2 C, P2 Hy dis C and WΦ C bind to different operators that vary in sequence, length, center-to-center spacing and location relative to the Pe promoter. The C repressors have different affinities to their operators and show different binding patterns. The binding capacity of the C proteins is not affected by point mutations of one or both half sites, which was unexpected since in vivo the proteins are unable to repress the operator where both half sites contain a point mutation. Deletions in the spacing between the half sites change the band pattern, but the repression capacity of WΦ C was unaffected. Only WΦ C is able to bind to just one half site. P2 C, P2 Hy dis C and WΦ C have the best affinity to their wild type operator, which was expected. Dissociation events of P2 and P2 Hy dis C were bi- and triphasic. Consequently, the kinetics and the nature of complexes formed show clearly that these three closely related bacteriophages have evolved in different directions. The N-terminal part of the C protein contains four α-helices, and the secondary structure predictions show two possible DNA-binding helices. According to the alanine scanning helix 3 might be the DNA-recognition helix since the mutations in this helix removed the repression capacity of the C protein in vivo.
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| 8. |
- Massad, Tariq, et al.
(författare)
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Crystal structure of the P2 C-repressor : a binder of nonpalindromic direct DNA repeats
- 2010
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 38:21, s. 7778-7790
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Tidskriftsartikel (refereegranskat)abstract
- As opposed to the vast majority of prokaryoticrepressors, the immunity repressor of temperateEscherichia coli phage P2 (C) recognizes nonpalindromicdirect repeats of DNA rather thaninverted repeats. We have determined the crystalstructure of P2 C at 1.8A ° . This constitutes the firststructure solved from the family of C proteins fromP2-like bacteriophages. The structure reveals thatthe P2 C protein forms a symmetric dimer orientedto bind the major groove of two consecutive turns ofthe DNA. Surprisingly, P2 C has great similarities tobinders of palindromic sequences. Nevertheless, thetwo identical DNA-binding helixes of the symmetricP2 C dimer have to bind different DNA sequences.Helix 3 is identified as the DNA-recognition motif inP2 C by alanine scanning and the importance for theindividual residues in DNA recognition is defined.A truncation mutant shows that the disorderedC-terminus is dispensable for repressor function.The short distance between the DNA-bindinghelices together with a possible interaction betweentwo P2 C dimers are proposed to be responsible forextensive bending of the DNA. The structure providesinsight into the mechanisms behind the mutants ofP2 C causing dimer disruption, temperature sensitivityand insensitivity to the P4 antirepressor.
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