Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Dec 8;32(21):6445-53.
doi: 10.1093/nar/gkh975. Print 2004.

DNA footprinting and biophysical characterization of the controller protein C.AhdI suggests the basis of a genetic switch

Affiliations

DNA footprinting and biophysical characterization of the controller protein C.AhdI suggests the basis of a genetic switch

S D Streeter et al. Nucleic Acids Res. .

Abstract

We have cloned and expressed the ahdIC gene of the AhdI restriction-modification system and have purified the resulting controller (C) protein to homogeneity. The protein sequence shows a HTH motif typical of that found in many transcriptional regulators. C.AhdI is found to form a homodimer of 16.7 kDa; sedimentation equilibrium experiments show that the dimer dissociates into monomers at low concentration, with a dissociation constant of 2.5 microM. DNase I and Exo III footprinting were used to determine the C.AhdI DNA-binding site, which is found approximately 30 bp upstream of the ahdIC operon. The intact homodimer binds cooperatively to a 35 bp fragment of DNA containing the C-protein binding site with a dissociation constant of 5-6 nM, as judged both by gel retardation analysis and by surface plasmon resonance, although in practice the affinity for DNA is dominated by protein dimerization as DNA binding by the monomer is negligible. The location of the C-operator upstream of both ahdIC and ahdIR suggests that C.AhdI may act as a positive regulator of the expression of both genes, and could act as a molecular switch that is critically dependent on the K(d) for the monomer-dimer equilibrium. Moreover, the structure and location of the C.AhdI binding site with respect to the putative -35 box preceding the C-gene suggests a possible mechanism for autoregulation of C.AhdI expression.

PubMed Disclaimer

Figures

Figure 1
Figure 1
(a) Arrangement of the AhdI R-M genes. The M and S genes, and the C and R genes are arranged convergently, with transcription in the direction shown. The two operons are separated by a central self-complementary region, depicted here as a hairpin. (b) Secondary structure prediction. The amino acid sequence of C.AhdI is given with the putative helix–turn–helix region underlined. Secondary structure predictions are shown from the program PSIPRED with associated reliability indices (Rel) on a scale of 1–10. Predicted helices are denoted as ‘H’.
Figure 2
Figure 2
PAGE showing stages in the purification of C.AhdI. Lane 1, molecular weight marker; lane 2, whole cell lysate; lane 3, ammonium sulfate pellet following resuspension and dialysis; lane 4, pooled heparin fractions; lane 5, resuspended protein precipitate; lane 6, pooled cation exchange fractions; lane 7, pooled size exclusion fractions; and lane 8, concentrated C.AhdI.
Figure 3
Figure 3
(a) Dynamic light scattering. The peak corresponds to a hydrodynamic radius of 1.97 nm, equivalent to a molecular mass of 16.5 kDa. Polydispersity is 10%. Protein concentration was 0.2 mg ml−1 (24 μM). (b) Static (Rayleigh) light scattering. A series of C.AhdI protein concentrations (0.099–0.493 mg ml−1) were analysed by static light scattering. The plot of residual intensity is shown in blue, (K)(C)/R90 is shown in red. The intercept on the (K)(C)/R90 axis gives 1/Mr, from which we obtain Mr = 16804 ± 376 Da.
Figure 4
Figure 4
Sedimentation equilibrium. (a) Weight-averaged Mr from sedimentation equilibrium runs for C.AhdI at a series of different concentrations, with associated errors from the fit. (b) The sedimentation equilibrium profile at 15 μM protein was fitted to a monomer–dimer equilibrium model, and residuals shown (× 10−3). The centrifuge was run at 21 000 r.p.m. for 13 h (20°C) and absorption monitored at 235 nm.
Figure 5
Figure 5
DNA footprinting of C.AhdI. (a) Exonuclease III footprinting. Cleavage sites are shown for the 5′-labelled top and bottom strands of the 35 bp DNA duplex (1 μM). The G + A ladder is shown to the left of each series, and concentration dependent cutting sites are indicated. The protein concentration range was 0–8 μM. (b) DNase I footprinting of C.AhdI. Cleavage sites are shown for the 5′-labelled top and bottom strands of the 60 bp DNA duplex (4 μM). The G + A ladder is shown to the left of each series, and concentration dependent cutting sites are indicated. The protein concentration range was 0–16 μM. (c) Summary of footprinting data. The sites of enhanced cutting by DNase I are shown as black arrows. The horizontal black bar shows the major regions of DNase I protection, along with secondary regions of protection (dotted line), thicker lines/arrows representing more prominent protection/enhancement. The principal ExoIII stop sites are indicated by open arrows, but there is a major additional stop site near the centre of the footprint which is prominent at intermediate protein concentrations (smaller arrow). The central 12 bp sequence which includes the symmetrical sequence is boxed. The putative −35 box sequence, TTGACT, is underlined and the −10 site, TGTAAT, italicized.
Figure 6
Figure 6
Electrophoretic mobility shift assay. (a) Protein samples of increasing concentration (0–1280 nM) were incubated with 80 nM DNA. The monomer (M) and dimer complexes (D) are indicated, together with the free DNA band (F). (b) The fraction bound was determined using ImageQuant software and the value plotted was the average of values obtained from two gels. The solid line shows the fitted curve for a monomer–dimer equilibrium model (see text) with K1 = 2.5 μM and K2 = 6.4 nM. For comparison, the dotted line shows the theoretical binding curve in the absence of dimer dissociation (K1 = 0 and K2 = 6.4 nM).
Figure 7
Figure 7
Surface plasmon resonance. C.AhdI samples of varying concentration (100, 200 and 300 nM) were injected at 30 μl min−1 and the binding curves were fitted to a 1:1 Langmuir binding model with mass transfer (dotted lines). A protein–DNA dissociation constant of 4.8 nM was calculated from the on-rate (4.8 × 106 M−1 s−1) and off-rate (0.023 s−1).
Figure 8
Figure 8
Analysis of C-protein DNA-binding sites. (a) Idealized symmetrical sequence and (b) consensus sequence (with fully conserved bases shown in bold) as proposed by Vijesurier et al. (5). (c) Binding region of C.AhdI: the location of the idealized (pentameric) symmetrical sequence for C.AhdI is shown in red; sequence identities with the consensus sequence of Vijesurier et al. are shown in bold, both within and outside of the symmetrical sequence. The proposed OL and OR sequences for AhdI are compared below, colour coding as above. (d) Comparison of boxes 1–4 in the C.AhdI binding site, with the reverse complementary sequence of boxes 2 and 4 shown to compare the dyad symmetry. Bases conserved in 3 out of 4 of the boxes are indicated in bold. Numbers shown on the right of each box refer to the number of base identities to the C.AhdI consensus sequence. (e) C.AhdI operator site, indicating the −35 sequence (green), the −10 sequence (purple) and the start codon for the C-protein (blue).

Similar articles

Cited by

References

    1. Tao T. and Blumenthal,R.M. (1992) Sequence and characterization of pvuIIR, the PvuII endonuclease gene, and of pvuIIC, its regulatory gene. J. Bacteriol., 174, 3395–3398. - PMC - PubMed
    1. Ives C.L., Nathan,P.D. and Brooks,J.E. (1992) Regulation of the BamHI restriction-modification system by a small intergenic open reading frame, bamHIC, in both Escherichia coli and Bacillus subtilis. J. Bacteriol., 174, 7194–7201. - PMC - PubMed
    1. Rimseliene R., Vaisvila,R. and Janulaitis,A. (1995) The eco72IC gene specifies a trans-acting factor which influences expression of both DNA methyltransferase and endonuclease from the Eco72I restriction-modification system. Gene, 157, 217–219. - PubMed
    1. Lubys A., Jurenaite,S. and Janulaitis,A. (1999) Structural organization and regulation of the plasmid-borne type II restriction-modification system Kpn2I from Klebsiella pneumoniae RFL2. Nucleic Acids Res., 27, 4228–4234. - PMC - PubMed
    1. Vijesurier R.M., Carlock,L., Blumenthal,R.M. and Dunbar,J.C. (2000) Role and mechanism of action of C·PvuII, a regulatory protein conserved among restriction-modification systems. J. Bacteriol., 182, 477–487. - PMC - PubMed

Publication types