Structural analysis of DNA binding by C.Csp231I, a member of a novel class of R-M controller proteins regulating gene expression.

In a wide variety of bacterial restriction-modification systems, a regulatory `controller' protein (or C-protein) is required for effective transcription of its own gene and for transcription of the endonuclease gene found on the same operon. We have recently turned our attention to a new class of controller proteins (exemplified by C.Csp231I) that have quite novel features, including a much larger DNA-binding site with an 18 bp (∼60 Å) spacer between the two palindromic DNA-binding sequences and a very different recognition sequence from the canonical GACT/AGTC. Using X-ray crystallography, the structure of the protein in complex with its 21 bp DNA-recognition sequence was solved to 1.8 Šresolution, and the molecular basis of sequence recognition in this class of proteins was elucidated. An unusual aspect of the promoter sequence is the extended spacer between the dimer binding sites, suggesting a novel interaction between the two C-protein dimers when bound to both recognition sites correctly spaced on the DNA. A U-bend model is proposed for this tetrameric complex, based on the results of gel-mobility assays, hydrodynamic analysis and the observation of key contacts at the interface between dimers in the crystal.

Structural analysis of DNA-protein complexes regulating the restriction-modification system Esp1396I.

The controller protein of the type II restriction-modification (RM) system Esp1396I binds to three distinct DNA operator sequences upstream of the methyltransferase and endonuclease genes in order to regulate their expression. Previous biophysical and crystallographic studies have shown molecular details of how the controller protein binds to the operator sites with very different affinities. Here, two protein-DNA co-crystal structures containing portions of unbound DNA from native operator sites are reported. The DNA in both complexes shows significant distortion in the region between the conserved symmetric sequences, similar to that of a DNA duplex when bound by the controller protein (C-protein), indicating that the naked DNA has an intrinsic tendency to bend when not bound to the C-protein. Moreover, the width of the major groove of the DNA adjacent to a bound C-protein dimer is observed to be significantly increased, supporting the idea that this DNA distortion contributes to the substantial cooperativity found when a second C-protein dimer binds to the operator to form the tetrameric repression complex.

The structural basis of differential DNA sequence recognition by restriction-modification controller proteins.

Controller (C) proteins regulate the expression of restriction-modification (RM) genes in a wide variety of RM systems. However, the RM system Esp1396I is of particular interest as the C protein regulates both the restriction endonuclease (R) gene and the methyltransferase (M) gene. The mechanism of this finely tuned genetic switch depends on differential binding affinities for the promoters controlling the R and M genes, which in turn depends on differential DNA sequence recognition and the ability to recognize dual symmetries. We report here the crystal structure of the C protein bound to the M promoter, and compare the binding affinities for each operator sequence by surface plasmon resonance. Comparison of the structure of the transcriptional repression complex at the M promoter with that of the transcriptional activation complex at the R promoter shows how subtle changes in protein-DNA interactions, underpinned by small conformational changes in the protein, can explain the molecular basis of differential regulation of gene expression.

Recognition of dual symmetry by the controller protein C.Esp1396I based on the structure of the transcriptional activation complex.

The controller protein C.Esp1396I regulates the timing of gene expression of the restriction-modification (RM) genes of the RM system Esp1396I. The molecular recognition of promoter sequences by such transcriptional regulators is poorly understood, in part because the DNA sequence motifs do not conform to a well-defined symmetry. We report here the crystal structure of the controller protein bound to a DNA operator site. The structure reveals how two different symmetries within the operator are simultaneously recognized by the homo-dimeric protein, underpinned by a conformational change in one of the protein subunits. The recognition of two different DNA symmetries through movement of a flexible loop in one of the protein subunits may represent a general mechanism for the recognition of pseudo-symmetric DNA sequences.

Structural analysis of a novel class of R-M controller proteins: C.Csp231I from Citrobacter sp. RFL231.

Controller proteins play a key role in the temporal regulation of gene expression in bacterial restriction-modification (R-M) systems and are important mediators of horizontal gene transfer. They form the basis of a highly cooperative, concentration-dependent genetic switch involved in both activation and repression of R-M genes. Here we present biophysical, biochemical, and high-resolution structural analysis of a novel class of controller proteins, exemplified by C.Csp231I. In contrast to all previously solved C-protein structures, each protein subunit has two extra helices at the C-terminus, which play a large part in maintaining the dimer interface. The DNA binding site of the protein is also novel, having largely AAAA tracts between the palindromic recognition half-sites, suggesting tight bending of the DNA. The protein structure shows an unusual positively charged surface that could form the basis for wrapping the DNA completely around the C-protein dimer.

Overexpression, purification and preliminary X-ray diffraction analysis of the controller protein C.Csp231I from Citrobacter sp. RFL231.

Restriction-modification controller proteins play an essential role in regulating the temporal expression of restriction-modification genes. The controller protein C.Csp231I represents a new class of controller proteins. The gene was sublconed to allow overexpression in Escherichia coli. The protein was purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 2.0 A resolution and belonged to space group P2(1). An electrophoretic mobility-shift assay provided evidence of strong binding of C.Csp231I to a sequence located upstream of the csp231IC start codon.

Structure of the restriction-modification controller protein C.Esp1396I.

The controller protein of the Esp1396I restriction-modification (R-M) system binds differentially to three distinct operator sequences upstream of the methyltransferase (M) and endonuclease (R) genes to regulate the timing of gene expression. The crystal structure of a complex of the protein with two adjacent operator DNA sequences has been reported; however, the structure of the free protein has not yet been determined. Here, the crystal structure of the free protein is reported, with seven dimers in the asymmetric unit. Two of the 14 monomers show an alternative conformation to the major conformer in which the side chains of residues 43-46 in the loop region flanking the DNA-recognition helix are displaced by up to 10 A. It is proposed that the adoption of these two conformational states may play a role in DNA-sequence promiscuity. The two alternative conformations are also found in the R35A mutant structure, which is otherwise identical to the native protein. Comparison of the free and bound protein structures shows a 1.4 A displacement of the recognition helices when the dimer is bound to its DNA target.

DNA structural deformations in the interaction of the controller protein C.AhdI with its operator sequence.

Controller proteins such as C.AhdI regulate the expression of bacterial restriction-modification genes, and ensure that methylation of the host DNA precedes restriction by delaying transcription of the endonuclease. The operator DNA sequence to which C.AhdI binds consists of two adjacent binding sites, O(L) and O(R). Binding of C.AhdI to O(L) and to O(L) + O(R) has been investigated by circular permutation DNA-bending assays and by circular dichroism (CD) spectroscopy. CD indicates considerable distortion to the DNA when bound by C.AhdI. Binding to one or two sites to form dimeric and tetrameric complexes increases the CD signal at 278 nm by 40 and 80% respectively, showing identical local distortion at both sites. In contrast, DNA-bending assays gave similar bend angles for both dimeric and tetrameric complexes (47 and 38 degrees, respectively). The relative orientation of C.AhdI dimers in the tetrameric complex and the structural role of the conserved Py-A-T sequences found at the centre of C-protein-binding sites are discussed.

Shape and subunit organisation of the DNA methyltransferase M.AhdI by small-angle neutron scattering.

Type I restriction-modification (R-M) systems encode multisubunit/multidomain enzymes. Two genes (M and S) are required to form the methyltransferase (MTase) that methylates a specific base within the recognition sequence and protects DNA from cleavage by the endonuclease. The DNA methyltransferase M.AhdI is a 170 kDa tetramer with the stoichiometry M(2)S(2) and has properties typical of a type I MTase. The M.AhdI enzyme has been prepared with deuterated S subunits, to allow contrast variation using small-angle neutron scattering (SANS) methods. The SANS data were collected in a number of (1)H:(2)H solvent contrasts to allow matching of one or other of the subunits in the multisubunit enzyme. The radius of gyration (R(g)) and maximum dimensions (D(max)) of the M subunits in situ in the multisubunit enzyme (50 A and 190 A, respectively) are close of those of the entire MTase (51 A and 190 A). In contrast, the S subunits in situ have experimentally determined values of R(g)=35 A and D(max)=110 A, indicating their more central location in the enzyme. Ab initio reconstruction methods yield a low-resolution structural model of the shape and subunit organization of M.AhdI, in which the Z-shaped structure of the S subunit dimer can be discerned. In contrast, the M subunits form a much more elongated and extended structure. The core of the MTase comprises the two S subunits and the globular regions of the two M subunits, with the extended portion of the M subunits most probably forming highly mobile regions at the outer extremities, which collapse around the DNA when the MTase binds.

Cooperative binding of the C.AhdI controller protein to the C/R promoter and its role in endonuclease gene expression.

The controller (C) proteins of a wide variety of restriction-modification (R-M) systems are thought to regulate expression of the endonuclease (R) gene by a genetic switch that ensures that methylation precedes endonuclease expression. Previous DNA footprinting experiments with C.AhdI have located the binding site upstream of the C and R genes in the AhdI R-M system, and the structure of C.AhdI has recently been determined. Here, we provide evidence that the binding site can accommodate either one or two dimers of C.AhdI in a concentration-dependent manner. The dimer binding site is adjacent to the -35 hexamer site required for the interaction with RNA polymerase (RNAP); however, co-operative binding of a second dimer blocks this site. Optimum DNA binding site sizes for dimer and tetramer formation were determined to be ca 21 bp and 34 bp, respectively. The stoichiometry and affinities of relevant DNA-protein complexes have been characterised by sedimentation velocity and EMSA using native and mutant promoter sequences. Molecular models of the dimer and tetramer complexes have been constructed that are consistent with the hydrodynamic data. Our results suggest a mechanism for both positive and negative regulation of endonuclease expression, whereby at moderate levels of C.AhdI, the protein binds to the promoter as a dimer and stimulates transcription by the interaction with RNAP. As the levels of C.AhdI increase further, binding of the second dimer competes with RNAP, thus down-regulating transcription of its own gene, and hence that of the endonuclease.

High-resolution crystal structure of the restriction-modification controller protein C.AhdI from Aeromonas hydrophila.

Restriction-modification (R-M) systems serve to protect the host bacterium from invading bacteriophage. The multi-component system includes a methyltransferase, which recognizes and methylates a specific DNA sequence, and an endonuclease which recognises the same sequence and cleaves within or close to this site. The endonuclease will only cleave DNA that is unmethylated at the specific site, thus host DNA is protected while non-host DNA is cleaved. However, following DNA replication, expression of the endonuclease must be delayed until the host DNA is appropriately methylated. In many R-M systems, this regulation is achieved at the transcriptional level via the controller protein, or C-protein. We have solved the first X-ray structure of an R-M controller protein, C.AhdI, to 1.69 A resolution using selenomethionine MAD. C.AhdI is part of a Type IIH R-M system from the pathogen Aeromonas hydrophila. The structure reveals an all-alpha protein that contains a classical helix-turn-helix (HTH) domain and can be assigned to the Xre family of transcriptional regulators. Unlike its monomeric structural homologues, an extended helix generates an interface that results in dimerisation of the free protein. The dimer is electrostatically polarised and a positively charged surface corresponds to the position of the DNA recognition helices of the HTH domain. Comparison with the structure of the lambda cI ternary complex suggests that C.AhdI activates transcription through direct contact with the sigma70 subunit of RNA polymerase.

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

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.

Crystallization and preliminary X-ray analysis of the controller protein C.AhdI from Aeromonas hydrophilia.

Single crystals of purified homodimeric controller protein from Aeromonas hydrophilia (C.AhdI) have been grown under several different conditions using vapour diffusion. X-ray diffraction data have been collected using synchrotron radiation from crystals of both the native and a selenomethionine (SeMet) derivative of the protein. The native crystal form belongs to space group P2(1) and data were collected to a resolution of 2.2 A. Two crystal forms of the SeMet protein have been obtained and were found to belong to space groups P1 and P2(1); data have been recorded to 2.0 and 1.7 A resolution, respectively, for the two crystal forms. Three-wavelength MAD data were collected to 1.7 A for the SeMet derivative crystal, which is isomorphous with the native P2(1) crystal.

Domain structure and subunit interactions in the type I DNA methyltransferase M.EcoR124I.

The type IC DNA methyltransferase M.EcoR124I is a trimeric enzyme of 162 kDa consisting of two modification subunits, HsdM, and a single specificity subunit, HsdS. Studies have been largely restricted to the HsdM subunit or to the intact methyltransferase since the HsdS subunit is insoluble when over-expressed independently of HsdM. Two soluble fragments of the HsdS subunit have been cloned, expressed and purified; a 25 kDa N-terminal fragment (S3) comprising the N-terminal target recognition domain together with the central conserved domain, and a 8.6 kDa fragment (S11) comprising the central conserved domain alone. Analytical ultracentrifugation shows that the S3 subunit exists principally as a dimer of 50 kDa. Gel retardation and competition assays show that both S3 and S11 are able to bind to HsdM, each with a subunit stoichiometry of 1:1. The tetrameric complex (S3/HsdM)(2) is required for effective DNA binding. Cooperative binding is observed and at low enzyme concentration, the multisubunit complex dissociates, leading to a loss of DNA binding activity. The (S3/HsdM)(2) complex is able to bind to both the EcoR124I DNA recognition sequence GAAN(6)RTCG and a symmetrical DNA sequence GAAN(7)TTC, but has a 30-fold higher affinity binding for the latter DNA sequence. Exonuclease III footprinting of the (S3/HsdM)(2) -DNA complex indicates that 29 nucleotides are protected on each strand, corresponding to a region 8 bp on both the 3' and 5' sides of the recognition sequence bound by the (S3/HsdM)(2) complex.

The DNA-binding domain of the gene regulatory protein AreA extends beyond the minimal zinc-finger region conserved between GATA proteins.

The AreA protein of Aspergillus nidulans regulates the activity of over 100 genes involved in the utilisation of nitrogen, and has a limited region of homology with the vertebrate family of GATA proteins around a zinc finger (Zf) motif. A 66 amino acid (a.a.) residue fragment (Zf(66)) corresponding to the zinc finger, a 91 a.a fragment (Zf(91)) containing an additional 25 a.a. at the C-terminus, and a much larger 728 a.a. sequence (3'EX) corresponding to the 3'exon have been over-expressed as fusion proteins in E. coli and purified. The DNA-protein complexes formed by these proteins have been examined by gel retardation analysis. The 91 a.a. protein forms a discrete shifted species with a GATA-containing DNA fragment with high affinity (K(d)=0.15 nM), whereas the 66 a.a. protein has very low ( approximately microM) affinity for the same sequence. The results show that the region of AreA required for high affinity DNA binding extends beyond the zinc finger motif that is homologous to GATA-1, requiring in addition a region within the 25 a.a. sequence C-terminal to the zinc finger. Using hydroxyl radical and ethylation interference footprinting, the minimal Zinc finger protein (Zf(66)) shows no appreciable interference effects whereas Zf(91) shows much stronger interference effects, identical to those of the larger protein. These effects extend over sequences up to two nucleotides either side of the GATA site, and indicate contacts additional to those observed in the three-dimensional structure of the complex of the minimal zinc-finger protein with DNA. We suggest that these additional contacts are responsible for the enhanced DNA binding affinity of the extended zinc-finger protein Zf(91).

Preferential binding of fd gene 5 protein to tetraplex nucleic acid structures.

The gene 5 protein of filamentous bacteriophage fd is a single-stranded DNA-binding protein that binds non-specifically to all single-stranded nucleic acid sequences, but in addition is capable of specific binding to the sequence d(GT(5)G(4)CT(4)C) and the RNA equivalent r(GU(5)G(4)CU(4)C), the latter interaction being important for translational repression. We show that this sequence preference arises from the formation of a tetraplex structure held together by a central block of G-quartets, the structure of which persists in the complex with gene 5 protein. Binding of gene 5 protein to the tetraplex leads to formation of a approximately 170 kDa nucleoprotein complex consisting of four oligonucleotide strands and eight gene 5 protein dimers, with a radius of gyration of 45 A and an overall maximum dimension of 120-130 A. A model of the complex is presented that is consistent with the data obtained. It is proposed that the G-quartet may act as a nucleation site for binding gene 5 protein to adjacent single-stranded regions, suggesting a novel mechanism for translational repression.