Base-specific Contacts Research Articles

An ancient protein-DNA interaction underlying metazoan sex determination.

DMRT transcription factors are deeply conserved regulators of metazoan sexual development. They share the DM DNA-binding domain, a unique intertwined double zinc-binding module followed by a C-terminal recognition helix, which binds a pseudopalindromic target DNA. Here we show that DMRT proteins use a unique binding interaction, inserting two adjacent antiparallel recognition helices into a widened DNA major groove to make base-specific contacts. Versatility in how specific base contacts are made allows human DMRT1 to use multiple DNA binding modes (tetramer, trimer and dimer). Chromatin immunoprecipitation with exonuclease treatment (ChIP-exo) indicates that multiple DNA binding modes also are used in vivo. We show that mutations affecting residues crucial for DNA recognition are associated with an intersex phenotype in flies and with male-to-female sex reversal in humans. Our results illuminate an ancient molecular interaction underlying much of metazoan sexual development.

Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5' splice site recognition.

U1 snRNP binds to the 5' exon-intron junction of pre-mRNA and thus plays a crucial role at an early stage of pre-mRNA splicing. We present two crystal structures of engineered U1 sub-structures, which together reveal at atomic resolution an almost complete network of protein-protein and RNA-protein interactions within U1 snRNP, and show how the 5' splice site of pre-mRNA is recognised by U1 snRNP. The zinc-finger of U1-C interacts with the duplex between pre-mRNA and the 5'-end of U1 snRNA. The binding of the RNA duplex is stabilized by hydrogen bonds and electrostatic interactions between U1-C and the RNA backbone around the splice junction but U1-C makes no base-specific contacts with pre-mRNA. The structure, together with RNA binding assays, shows that the selection of 5'-splice site nucleotides by U1 snRNP is achieved predominantly through basepairing with U1 snRNA whilst U1-C fine-tunes relative affinities of mismatched 5'-splice sites.

DNA Recognition by a σ54 Transcriptional Activator from Aquifex aeolicus

Transcription initiation by bacterial σ54-polymerase requires the action of a transcriptional activator protein. Activators bind sequence-specifically upstream of the transcription initiation site via a DNA-binding domain (DBD). The structurally characterized DBDs from activators all belong to the Fis (factor for inversion stimulation) family of helix–turn–helix DNA-binding proteins. We report here structures of the free and DNA-bound forms of the DBD of NtrC4 (4DBD) from Aquifex aeolicus, a member of the NtrC family of σ54 activators. Two NtrC4-binding sites were identified upstream (−145 and −85bp) from the start of the lpxC gene, which is responsible for the first committed step in lipid A biosynthesis. This is the first experimental evidence for σ54 regulation in lpxC expression. 4DBD was crystallized both without DNA and in complex with the −145-binding site. The structures, together with biochemical data, indicate that NtrC4 binds to DNA in a manner that is similar to that of its close homolog, Fis. The greater sequence specificity for the binding of 4DBD relative to Fis seems to arise from a larger number of base-specific contacts contributing to affinity than for Fis.

Pentatricopeptide Repeat Proteins in Plants

Pentatricopeptide repeat (PPR) proteins constitute one of the largest protein families in land plants, with more than 400 members in most species. Over the past decade, much has been learned about the molecular functions of these proteins, where they act in the cell, and what physiological roles they play during plant growth and development. A typical PPR protein is targeted to mitochondria or chloroplasts, binds one or several organellar transcripts, and influences their expression by altering RNA sequence, turnover, processing, or translation. Their combined action has profound effects on organelle biogenesis and function and, consequently, on photosynthesis, respiration, plant development, and environmental responses. Recent breakthroughs in understanding how PPR proteins recognize RNA sequences through modular base-specific contacts will help match proteins to potential binding sites and provide a pathway toward designing synthetic RNA-binding proteins aimed at desired targets.

ExsA and LcrF Recognize Similar Consensus Binding Sites, but Differences in Their Oligomeric State Influence Interactions with Promoter DNA

ExsA activates type III secretion system (T3SS) gene expression in Pseudomonas aeruginosa and is a member of the AraC family of transcriptional regulators. AraC proteins contain two helix-turn-helix (HTH) DNA binding motifs. One helix from each HTH motif inserts into the major groove of the DNA to make base-specific contacts with the promoter region. The amino acids that comprise the HTH motifs of ExsA are nearly identical to those in LcrF/VirF, the activators of T3SS gene expression in the pathogenic yersiniae. In this study, we tested the hypothesis that ExsA/LcrF/VirF recognize a common nucleotide sequence. We report that Yersinia pestis LcrF binds to and activates transcription of ExsA-dependent promoters in P. aeruginosa and that plasmid-expressed ExsA complements a Y. pestis lcrF mutant for T3SS gene expression. Mutations that disrupt the ExsA consensus binding sites in both P. aeruginosa and Y. pestis T3SS promoters prevent activation by ExsA and LcrF. Our combined data demonstrate that ExsA and LcrF recognize a common nucleotide sequence. Nevertheless, the DNA binding properties of ExsA and LcrF are distinct. Whereas two ExsA monomers are sequentially recruited to the promoter region, LcrF binds to promoter DNA as a preformed dimer and has a higher capacity to bend DNA. An LcrF mutant defective for dimerization bound promoter DNA with properties similar to ExsA. Finally, we demonstrate that the activators of T3SS gene expression from Photorhabdus luminescens, Aeromonas hydrophila, and Vibrio parahaemolyticus are also sensitive to mutations that disrupt the ExsA consensus binding site.

Nucleotide-Specific Recognition of Iron-Responsive Elements by Iron Regulatory Protein 1

IRP1 [iron regulatory protein (IRP) 1] is a bifunctional protein with mutually exclusive end-states. In one mode of operation, IRP1 binds iron-responsive element (IRE) stem–loops in messenger RNAs encoding proteins of iron metabolism to control their rate of translation. In its other mode, IRP1 serves as cytoplasmic aconitase to correlate iron availability with the energy and oxidative stress status of the cell. IRP1/IRE binding occurs through two separate interfaces, which together contribute about two-dozen hydrogen bonds. Five amino acids make base-specific contacts and are expected to contribute significantly to binding affinity and specificity of this protein:RNA interaction. In this mutagenesis study, each of the five base-specific amino acids was changed to alter binding at each site. Analysis of IRE binding affinity and translational repression activity of the resulting IRP1 mutants showed that four of the five contact points contribute uniquely to the overall binding affinity of the IRP1:IRE interaction, while one site was found to be unimportant. The stronger-than-expected effect on binding affinity of mutations at Lys379 and Ser681, residues that make contact with the conserved nucleotides G16 and C8, respectively, identified them as particularly critical for providing specificity and stability to IRP1:IRE complex formation. We also show that even though the base-specific RNA-binding residues are not part of the aconitase active site, their substitutions can affect the aconitase activity of holo-IRP1, positively or negatively.

The transcription factor AmrZ utilizes multiple DNA binding modes to recognize activator and repressor sequences of Pseudomonas aeruginosa virulence genes.

AmrZ, a member of the Ribbon-Helix-Helix family of DNA binding proteins, functions as both a transcriptional activator and repressor of multiple genes encoding Pseudomonas aeruginosa virulence factors. The expression of these virulence factors leads to chronic and sustained infections associated with worsening prognosis. In this study, we present the X-ray crystal structure of AmrZ in complex with DNA containing the repressor site, amrZ1. Binding of AmrZ to this site leads to auto-repression. AmrZ binds this DNA sequence as a dimer-of-dimers, and makes specific base contacts to two half sites, separated by a five base pair linker region. Analysis of the linker region shows a narrowing of the minor groove, causing significant distortions. AmrZ binding assays utilizing sequences containing variations in this linker region reveals that secondary structure of the DNA, conferred by the sequence of this region, is an important determinant in binding affinity. The results from these experiments allow for the creation of a model where both intrinsic structure of the DNA and specific nucleotide recognition are absolutely necessary for binding of the protein. We also examined AmrZ binding to the algD promoter, which results in activation of the alginate exopolysaccharide biosynthetic operon, and found the protein utilizes different interactions with this site. Finally, we tested the in vivo effects of this differential binding by switching the AmrZ binding site at algD, where it acts as an activator, for a repressor binding sequence and show that differences in binding alone do not affect transcriptional regulation.

Structural basis for sequence-specific recognition of DNA by TAL effectors.

TAL (transcription activator-like) effectors, secreted by phytopathogenic bacteria, recognize host DNA sequences through a central domain of tandem repeats. Each repeat comprises 33 to 35 conserved amino acids and targets a specific base pair by using two hypervariable residues [known as repeat variable diresidues (RVDs)] at positions 12 and 13. Here, we report the crystal structures of an 11.5-repeat TAL effector in both DNA-free and DNA-bound states. Each TAL repeat comprises two helices connected by a short RVD-containing loop. The 11.5 repeats form a right-handed, superhelical structure that tracks along the sense strand of DNA duplex, with RVDs contacting the major groove. The 12th residue stabilizes the RVD loop, whereas the 13th residue makes a base-specific contact. Understanding DNA recognition by TAL effectors may facilitate rational design of DNA-binding proteins with biotechnological applications.

Molecular determinants of origin discrimination by Orc1 initiators in archaea

Unlike bacteria, many eukaryotes initiate DNA replication from genomic sites that lack apparent sequence conservation. These loci are identified and bound by the origin recognition complex (ORC), and subsequently activated by a cascade of events that includes recruitment of an additional factor, Cdc6. Archaeal organisms generally possess one or more Orc1/Cdc6 homologs, belonging to the Initiator clade of ATPases associated with various cellular activities (AAA+) superfamily; however, these proteins recognize specific sequences within replication origins. Atomic resolution studies have shown that archaeal Orc1 proteins contact double-stranded DNA through an N-terminal AAA+ domain and a C-terminal winged-helix domain (WHD), but use remarkably few base-specific contacts. To investigate the biochemical effects of these associations, we mutated the DNA-interacting elements of the Orc1-1 and Orc1-3 paralogs from the archaeon Sulfolobus solfataricus, and tested their effect on origin binding and deformation. We find that the AAA+ domain has an unpredicted role in controlling the sequence selectivity of DNA binding, despite an absence of base-specific contacts to this region. Our results show that both the WHD and ATPase region influence origin recognition by Orc1/Cdc6, and suggest that not only DNA sequence, but also local DNA structure help define archaeal initiator binding sites.

Selection patterns on restorer-like genes reveal a conflict between nuclear and mitochondrial genomes throughout angiosperm evolution

Eukaryotic cells have harbored mitochondria for at least 1.5 billion years in an apparently mutually beneficial symbiosis. Studies on the agronomically important crop trait cytoplasmic male sterility (CMS) have suggested the semblance of a host-parasite relationship between the nuclear and mitochondrial genomes, but molecular evidence for this is lacking. Key players in CMS systems are the fertility restorer (Rf) genes required for the development of a functional male gametophyte in plants carrying a mitochondrial CMS gene. In the majority of cases, Rf genes encode pentatricopeptide repeat (PPR) proteins. We show that most angiosperms for which extensive genomic sequence data exist contain multiple PPR genes related to Rf genes. These Rf-like genes show a number of characteristic features compared with other PPR genes, including chromosomal clustering and unique patterns of evolution, notably high rates of nonsynonymous to synonymous substitutions, suggesting diversifying selection. The highest probabilities of diversifying selection were seen for amino acid residues 1, 3, and 6 within the PPR motif. PPR proteins are involved in RNA processing, and mapping the selection data to a predicted consensus structure of an array of PPR motifs suggests that these residues are likely to form base-specific contacts to the RNA ligand. We suggest that the selection patterns on Rf-like genes reveal a molecular "arms-race" between the nuclear and mitochondrial genomes that has persisted throughout most of the evolutionary history of angiosperms.

In VivoRecognition of thefecA3Target Promoter byHelicobacter pyloriNikR

In Helicobacter pylori, the transcriptional regulator HpNikR represses transcription of the fecA3 gene by binding to two adjacent operators spanning a region of almost 80 nucleotides along the fecA3 promoter in a nickel-dependent manner. By employing hydroxyl radical footprinting, we mapped the protected nucleotides within each operator. Three short sequences rich in A and T nucleotides were identified within each operator, comprising just 24 bases for both operators, with 4 or 5 protected bases interspaced by 4 to 7 free nucleotides, with no center of symmetry. Base substitutions at any site strongly reduced the affinity of HpNikR for the operators and also affected the stability of the DNA-protein complex, when the promoter-regulator interaction was analyzed in vitro. The effect of these substitutions was remarkably different when transcription of the mutant promoters was analyzed in vivo. Base changes introduced at the farthest subsites impaired the HpNikR-dependent repression, with the mutations closer to +1 completely abolishing the repression, the more distal one still allowing almost 50% of transcription, and the mutations in the middle being ineffective. The data presented here show that HpNikR may first select its targets by identifying sequences within the previously defined consensus and subsequently establish base-specific contacts to firmly bind DNA. In particular, HpNikR seems to interact in an asymmetric mode with the fecA3 target to repress its transcription.

Structure of the human FOXO4-DBD–DNA complex at 1.9 Å resolution reveals new details of FOXO binding to the DNA

FOXO4 is a member of the FOXO subgroup of forkhead transcription factors that constitute key components of a conserved signalling pathway that connects growth and stress signals to transcriptional control. Here, the 1.9 Å resolution crystal structure of the DNA-binding domain of human FOXO4 (FOXO4-DBD) bound to a 13 bp DNA duplex containing a FOXO consensus binding sequence is reported. The structure shows a similar recognition of the core sequence as has been shown for two other FOXO proteins. Helix H3 is docked into the major groove and provides all of the base-specific contacts, while the N-terminus and wing W1 make additional contacts with the phosphate groups of DNA. In contrast to other FOXO-DBD-DNA structures, the loop between helices H2 and H3 has a different conformation and participates in DNA binding. In addition, the structure of the FOXO4-DBD-DNA complex suggests that both direct water-DNA base contacts and the unique water-network interactions contribute to FOXO-DBD binding to the DNA in a sequence-specific manner.

The PAX3 Paired Domain and Homeodomain Function as a Single Binding Module In Vivo to Regulate Subnuclear Localization and Mobility by a Mechanism That Requires Base-Specific Recognition

The transcription factor PAX3 is essential for myogenesis and neural crest development, and is one of several genes mutated in human Waardenburg syndrome. Analysis of disease-causing missense mutations in PAX3 has established the interdependence of its two DNA-binding domains, the paired domain (PD) and the homeodomain (HD), as well as defects in localization and mobility. Paradoxically, mutants that retained DNA binding activity exhibited the greatest defects in localization and mobility, regardless of the domain in which they reside. In the present study, structure–function analyses were used to determine the mechanistic basis of this effect. In the context of the isolated DNA-binding domains, HD mutants adopted an increase in mobility proportional to their loss in DNA binding, while PD mutants continued to display the inverse relationship observed in the full-length protein. At the structural level, this reflected an unexpected dependence on base-specific contacts in the PD, whereas HD mobility was more severely affected by loss of backbone contacts, as has been observed with other DNA-binding proteins. This requires that the HD switch to a base-specific mode in the full-length protein. Moreover, both domains underwent substantial reduction in mobility and altered localization when in a contiguous polypeptide with the endogenous linker segment. Notably, although the HD conferred localization to heterochromatin, this activity was masked when linked to the PD, despite the absence of determinants for subnuclear compartmentalization in the PD or linker. Last, the propensity for PAX3 heterochromatin localization was modulated by sequences at the amino and carboxy termini, supporting a model in which alternate conformations lead to unmasking of the HD. These data indicate that the PD and the HD functionally interact in vivo and behave as a single binding module whose mobility and localization are dependent on sequence-specific contacts.

Argonaute MID domain takes centre stage

Two recent papers, one in EMBO reports and one in Nature give us the first eukaryotic structures of Argonaute MID domains; providing a structural basis for the 5′-nucleotide recognition of the guide strand and a possible explanation for the allosteric regulation of RNA binding.

The protein–DNA contacts in RutR·carAB operator complexes

Pyrimidine-specific regulation of the upstream carP1 promoter of the carbamoylphosphate synthase operon of Escherichia coli requires numerous trans-acting factors: the allosteric transcription regulator RutR, the nucleoid-associated protein integration host factor, and the trigger enzymes aminopeptidase A and PyrH (UMP-kinase). RutR, a TetR family member, binds far upstream of carP1. Here, we establish a high-resolution contact map of RutR•carP1 complexes for backbone and base-specific contacts, analyze DNA bending, determine the DNA sequence specificity of RutR binding by saturation mutagenesis, demonstrate that uracil but not thymine is the physiologically relevant ligand that inhibits the DNA binding capacity of RutR and build a model of the RutR·operator DNA complex based on the crystal structures of RutR and of the DNA-bound family member QacR. Finally, we test the validity of this model with site-directed mutagenesis of the helix–turn–helix DNA binding motif and in vitro binding studies with the cognate purified mutant RutR proteins.

Structural Determinants of DNA Binding by a P. falciparum ApiAP2 Transcriptional Regulator

Putative transcription factors have only recently been identified in the Plasmodium spp., with the major family of regulators comprising the Apicomplexan Apetala2 (AP2) proteins. To better understand the DNA-binding mechanisms of these transcriptional regulators, we characterized the structure and in vitro function of an AP2 DNA-binding domain from a prototypical Apicomplexan AP2 protein, PF14_0633 from Plasmodium falciparum. The X-ray crystal structure of the PF14_0633 AP2 domain bound to DNA reveals a β-sheet fold that binds the DNA major groove through base-specific and backbone contacts; a prominent α-helix supports the β-sheet structure. Substitution of predicted DNA-binding residues with alanine weakened or eliminated DNA binding in solution. In contrast to plant AP2 domains, the PF14_0633 AP2 domain dimerizes upon binding to DNA through a domain-swapping mechanism in which the α-helices of the AP2 domains pack against the β-sheets of the dimer mates. DNA-induced dimerization of PF14_0633 may be important for tethering two distal DNA loci together in the nucleus and/or for inducing functional rearrangements of its domains to facilitate transcriptional regulation. Consistent with a multisite binding mode, at least two copies of the consensus sequence recognized by PF14_0633 are present upstream of a previously identified group of sporozoite-stage genes. Taken together, these findings illustrate how Plasmodium has adapted the AP2 DNA-binding domain for genome-wide transcriptional regulation.

Water, Shape Recognition, Salt Bridges, and Cation–Pi Interactions Differentiate Peptide Recognition of the HIV Rev-Responsive Element

Recognition of the human immunodeficiency virus Rev-responsive element (RRE) RNA by the Rev protein is an essential step in the viral life cycle. Formation of the Rev–RRE complex signals nucleocytoplasmic export of unspliced and partially spliced viral RNA. Essential components of the complex have been localized to a minimal arginine-rich Rev peptide and stem IIB of RRE. In vitro selection studies have identified a synthetic peptide known as RSG 1.2 that binds with better specificity and affinity to RRE than the Rev peptide. NMR structures of both peptide–RNA complexes of Rev and RSG 1.2 bound to RRE stem IIB have been solved and reveal gross structural differences between the two bound complexes. Molecular dynamics simulations of the Rev and RSG 1.2 peptides in complex with RRE stem IIB have been simulated to better understand on an atomic level how two arginine-rich peptides of similar length recognize the same sequence of RNA with such different structural motifs. While the Rev peptide employs some base-specific hydrogen bonding for recognition of RRE, shape recognition, through contact with the sugar-phosphate backbone, and cation–pi interactions are also important. Molecular dynamics simulations suggest that RSG 1.2 binds more tightly to the RRE sequence than Rev by forming more base-specific contacts, using water to mediate peptide–RNA contacts, and is held in place by a strong salt bridge network spanning the major groove of the RNA.

Mutagenesis and Homology Modeling of the Tn21 Integron Integrase IntI1

Horizontal DNA transfer between bacteria is widespread and a major cause of antibiotic resistance. For logistic reasons, single or combined genes are shuttled between vectors such as plasmids and bacterial chromosomes. Special elements termed integrons operate in such shuttling and are therefore vital for horizontal gene transfer. Shorter elements carrying genes, cassettes, are integrated in the integrons, or excised from them, by virtue of a recombination site, attC, positioned in the 3' end of each unit. It is a remarkable and possibly restricting elementary feature of attC that it must be single-stranded while the partner target site, attI, may be double-stranded. The integron integrases belong to the tyrosine recombinase family, and this work reports mutations of the integrase IntI1 from transposon Tn21, chosen within a well-conserved region characteristic of the integron integrases. The mutated proteins were tested for binding to a bottom strand of an attC substrate, by using an electrophoresis mobility shift assay. To aid in interpreting the results, a homology model was constructed on the basis of the crystal structure of integron integrase VchIntIA from Vibrio cholerae bound to its cognate attC substrate VCRbs. The local stability and hydrogen bonding network of key domains of the modeled structure were further examined using molecular dynamics simulations. The homology model allowed us to interpret the roles of several amino acid residues, four of which were clearly binding assay responsive upon mutagenesis. Notably, we also observed features indicating that IntI1 may be more prone to base-specific contacts with VCRbs than VchIntIA.

Structural basis for LEAFY floral switch function and similarity with helix-turn-helix proteins

The LEAFY (LFY) protein is a key regulator of flower development in angiosperms. Its gradually increased expression governs the sharp floral transition, and LFY subsequently controls the patterning of flower meristems by inducing the expression of floral homeotic genes. Despite a wealth of genetic data, how LFY functions at the molecular level is poorly understood. Here, we report crystal structures for the DNA-binding domain of Arabidopsis thaliana LFY bound to two target promoter elements. LFY adopts a novel seven-helix fold that binds DNA as a cooperative dimer, forming base-specific contacts in both the major and minor grooves. Cooperativity is mediated by two basic residues and plausibly accounts for LFY's effectiveness in triggering sharp developmental transitions. Our structure reveals an unexpected similarity between LFY and helix-turn-helix proteins, including homeodomain proteins known to regulate morphogenesis in higher eukaryotes. The appearance of flowering plants has been linked to the molecular evolution of LFY. Our study provides a unique framework to elucidate the molecular mechanisms underlying floral development and the evolutionary history of flowering plants.

Copine-I represses NF-κB transcription by endoproteolysis of p65

Nuclear factor-kappaB (NF-kappaB) is a dynamic transcription factor that regulates important biological processes involved in cancer initiation and progression. Identifying regulators that control the half-life of NF-kappaB is important to understanding molecular processes that control the duration of transcriptional responses. In this study we identify copine-I, a calcium phospholipid-binding protein, as a novel repressor that physically interacts with p65 to inhibit NF-kappaB transcription. Knockdown of copine-I by siRNA increases tumor necrosis factor alpha-stimulated NF-kappaB transcription, while copine-I expression blocks endogenous transcription. Copine-I abolishes NF-kappaB transcription by inducing endoprotease processing of the N-terminus of p65, a process antagonized by IkappaB alpha. Copine-I stimulates endoproteolysis of p65 within a conserved region that is required for base-specific contact with DNA. p65 proteins lacking the N-terminus fail to bind to DNA and act as dominant-negative molecules that inhibit NF-kappaB transcription. Our work provides evidence that copine-I regulates the half-life of NF-kappaB transcriptional responses through a novel mechanism that involves endoproteolysis of the p65 protein.