Molecular Studies of Three Coliphage Repressor Proteins P2 C, P2 Hy dis C and Wphi C : Kinetics, Oligomeric States and Structural Studies

University dissertation from Stockholm : Institutionen för genetik, mikrobiologi och toxikologi

Abstract: P2, P2 Hy dis and W? 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 W? 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 W? C was unaffected. Only W? C is able to bind to just one half site. P2 C, P2 Hy dis C and W? 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 ?-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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