Determination of the DNA-binding kinetics of three related but heteroimmune bacteriophage repressors using EMSA and SPR analysis

Abstract

Bacteriophages P2, P2 Hy dis and WPhi are very similar but heteroimmune Escherichia coli phages. The structural genes show over 96% identity, but the repressors show between 43 and 63% identities. Furthermore, the operators, which contain two directly repeated sequences, vary in sequence, length, location relative to the promoter and spacing between the direct repeats. We have compared the in vivo effects of the wild type and mutated operators on gene expression with the complexes formed between the repressors and their wild type or mutated operators using electrophoretic mobility shift assay (EMSA), and real-time kinetics of the protein-DNA interactions using surface plasmon resonance (SPR) analysis. Using EMSA, the repressors formed different protein-DNA complexes, and only WPhi was significantly affected by point mutations. However, SPR analysis showed a reduced association rate constant and an increased dissociation rate constant for P2 and WPhi operator mutants. The association rate constants of P2 Hy dis was too fast to be determined. The P2 Hy dis dissociation response curves were shown to be triphasic, while both P2 and WPhi C were biphasic. Thus, the kinetics of complex formation and the nature of the complexes formed differ extensively between these very closely related phages.

Figure 1.

A comparison of the amino acid sequences of the C proteins and organization of the promoter–operator regions of P2, P2 Hy dis and Wϕ. (A) The amino acid sequence of the C proteins and the predicted secondary structure. Amino acids conserved among all three proteins are shaded in dark gray; amino acids conserved among two proteins are shaded in light gray. (B) DNA sequences of the early promoter–operators of P2, P2 Hy dis and Wϕ. The locations of the −10 and −35 regions are underlined. The transcriptional start sites are indicated by bent arrows. The operator half-sites are shaded in light gray, and the initiation codons are shaded in dark gray. The center-to-center distances between the half-sites are indicated in parenthesis. (C) The DNA sequences of the wt and mutated operators used in this work. The mutated nucleotide are shaded in gray.

Figure 2.

CAT assays showing the effects of the mutations in the direct repeats of P2 (A) and P2 Hy dis (B). In both cases, lane 1 is a control showing the strength of the Pe promoter in absence of the C repressor, and this CAT activity was set to 1. Lane 4 in B represents the operator where the O2 repeat extended with 2 nt corresponding to the O1 repeat. The relative CAT activity labeled 0, indicates that no activity could be detected in the phosphor imager analysis.

Figure 3.

Binding of the C proteins to their cognate operators analyzed by electro mobility shifts. The line above the respective autoradiograph shows the labeled DNA fragment used. The bold line represents DNA originating from the phage, and the thin line originates from the vector. The arrows represent the operator half-sites. The numbers below the line indicate the distance between the half-sites. (A). P2 wild-type operator (lanes 1–5) operator with a point mutation in O1 (lanes 6–10) and with a point mutation in both O1 and O2 (lanes 11–15). The P2 C protein is added in increasing amounts as indicated (18.4–373 nM). (B) P2 Hy dis wild-type operator (lanes 1–5) operator with a point mutation in O1 (lanes 6–10) and with a point mutation in both O1 and O2 (lanes 11–15). The P2 Hy dis C protein is added in increasing amounts as indicated (0.2–52 nM). (C)Wϕ wild-type operator (lanes 1–5), operator with a point mutation in O1 (lanes 6–10) and with a point mutation in both O1 and O2 (lanes 11–15). The Wϕ C protein is added in increasing amounts as indicated (0.1–2.2 nM).

Figure 4.

Titrations of complex formation between the C proteins and their respective wild-type operator, where the DNA concentration was constant and the protein concentration varied. The complex formation was determined using EMSA as the percent labeled DNA that was shifted out of total DNA loaded. (A) P2 C (0.03–1.3 nM). (B) P2 Hy dis C (2.1–13.8 nM). (C) Wϕ C (0.1–2.2 nM). Note that the protein concentration is in log scale and that only the P2 Hy dis C calculations are based on the experiment shown in Figure 3. The calculations are derived from titrations performed with the protein purifications having the highest specific activity.

Figure 5.

A comparison of the influence of the flow rate on the DNA binding of the C proteins to the respective wild-type operator in SPR analysis. (A). P2 C (B) P2 Hy dis C and (C). Wϕ C. The protein concentration was 23 pM. The injection time with Wϕ C at 30 μl/min was slightly longer compared with the injection time at 2 μl/min.

Figure 6.

Sensorgrams showing the influence of protein concentration on the SPR signals using wild-type operators. (A) P2 C (B) P2 Hy dis C and (C) Wϕ C.