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1.  The L7Ae protein binds to two kink-turns in the Pyrococcus furiosus RNase P RNA 
Nucleic Acids Research  2014;42(21):13328-13338.
The RNA-binding protein L7Ae, known for its role in translation (as part of ribosomes) and RNA modification (as part of sn/oRNPs), has also been identified as a subunit of archaeal RNase P, a ribonucleoprotein complex that employs an RNA catalyst for the Mg2+-dependent 5′ maturation of tRNAs. To better understand the assembly and catalysis of archaeal RNase P, we used a site-specific hydroxyl radical-mediated footprinting strategy to pinpoint the binding sites of Pyrococcus furiosus (Pfu) L7Ae on its cognate RNase P RNA (RPR). L7Ae derivatives with single-Cys substitutions at residues in the predicted RNA-binding interface (K42C/C71V, R46C/C71V, V95C/C71V) were modified with an iron complex of EDTA-2-aminoethyl 2-pyridyl disulfide. Upon addition of hydrogen peroxide and ascorbate, these L7Ae-tethered nucleases were expected to cleave the RPR at nucleotides proximal to the EDTA-Fe–modified residues. Indeed, footprinting experiments with an enzyme assembled with the Pfu RPR and five protein cofactors (POP5, RPP21, RPP29, RPP30 and L7Ae–EDTA-Fe) revealed specific RNA cleavages, localizing the binding sites of L7Ae to the RPR's catalytic and specificity domains. These results support the presence of two kink-turns, the structural motifs recognized by L7Ae, in distinct functional domains of the RPR and suggest testable mechanisms by which L7Ae contributes to RNase P catalysis.
PMCID: PMC4245976  PMID: 25361963
2.  Homotropic Cooperativity from the Activation Pathway of the Allosteric Ligand-Responsive Regulatory Protein TRAP† 
Biochemistry  2013;52(49):8855-8865.
The trp RNA-binding Attenuation Protein (TRAP) assembles into an 11-fold symmetric ring that regulates transcription and translation of trp-mRNA in bacilli via heterotropic allosteric activation by the amino acid tryptophan (Trp). Whereas nuclear magnetic resonance studies have revealed that Trp-induced activation coincides with both μs-ms rigidification and local structural changes in TRAP, the pathway of binding of the 11 Trp ligands to the TRAP ring remains unclear. Moreover, because each of eleven bound Trp molecules is completely surrounded by protein, its release requires flexibility of Trp-bound (holo) TRAP. Here, we used stopped-flow fluorescence to study the kinetics of Trp binding by Bacillus stearothermophilus TRAP over a range of temperatures and we observed well-separated kinetic steps. These data were analyzed using non-linear least-squares fitting of several two- and three-step models. We found that a model with two binding steps best describes the data, although the structural equivalence of the binding sites in TRAP implies a fundamental change in the time-dependent structure of the TRAP rings upon Trp binding. Application of the two binding step model reveals that Trp binding is much slower than the diffusion limit, suggesting a gating mechanism that depends on the dynamics of apo TRAP. These data also reveal that Trp dissociation from the second binding mode is much slower than after the first Trp binding mode, revealing insight into the mechanism for positive homotropic allostery, or cooperativity. Temperature dependent analyses reveal that both binding modes imbue increases in bondedness and order toward a more compressed active state. These results provide insight into mechanisms of cooperative TRAP activation, and underscore the importance of protein dynamics for ligand binding, ligand release, protein activation, and allostery.
PMCID: PMC3946311  PMID: 24224873
3.  Thermodynamics of Coupled Folding in the Interaction of Archaeal RNase P Proteins RPP21 and RPP29 
Biochemistry  2012;51(4):926-935.
We have used isothermal titration calorimetry (ITC) to identify and describe binding-coupled equilibria in the interaction between two protein subunits of archaeal ribonuclease P (RNase P). In all three domains of life, RNase P is a ribonucleoprotein complex that is primarily responsible for catalyzing the Mg2+-dependent cleavage of the 5′ leader sequence of precursor tRNAs during tRNA maturation. In archaea, RNase P has been shown to be composed of one catalytic RNA and up to five proteins, four of which associate in the absence of RNA as two functional heterodimers, POP5-RPP30 and RPP21-RPP29. NMR studies of the Pyrococcus furiosus RPP21 and RPP29 proteins in their free and complexed states provided evidence for significant protein folding upon binding. ITC experiments were performed over a range of temperatures, ionic strengths, pH values and in buffers with varying ionization potential, and with a folding-deficient RPP21 point mutant. These experiments revealed a negative heat capacity change (ΔCp), nearly twice that predicted from surface accessibility calculations, a strong salt dependence to the interaction and proton release at neutral pH, but a small net contribution from these to the excess ΔCp. We considered potential contributions from protein folding and burial of interfacial water molecules based on structural and spectroscopic data. We conclude that binding-coupled protein folding is likely responsible for a significant portion of the excess ΔCp. These findings provide novel structural-thermodynamic insights into coupled equilibria that enable specificity in macromolecular assemblies.
PMCID: PMC3964601  PMID: 22243443
4.  Bimodal high-affinity association of Brd4 with murine leukemia virus integrase and mononucleosomes 
Nucleic Acids Research  2014;42(8):4868-4881.
The importance of understanding the molecular mechanisms of murine leukemia virus (MLV) integration into host chromatin is highlighted by the development of MLV-based vectors for human gene-therapy. We have recently identified BET proteins (Brd2, 3 and 4) as the main cellular binding partners of MLV integrase (IN) and demonstrated their significance for effective MLV integration at transcription start sites. Here we show that recombinant Brd4, a representative of the three BET proteins, establishes complementary high-affinity interactions with MLV IN and mononucleosomes (MNs). Brd4(1–720) but not its N- or C-terminal fragments effectively stimulate MLV IN strand transfer activities in vitro. Mass spectrometry- and NMR-based approaches have enabled us to map key interacting interfaces between the C-terminal domain of BRD4 and the C-terminal tail of MLV IN. Additionally, the N-terminal fragment of Brd4 binds to both DNA and acetylated histone peptides, allowing it to bind tightly to MNs. Comparative analyses of the distributions of various histone marks along chromatin revealed significant positive correlations between H3- and H4-acetylated histones, BET protein-binding sites and MLV-integration sites. Our findings reveal a bimodal mechanism for BET protein-mediated MLV integration into select chromatin locations.
PMCID: PMC4005663  PMID: 24520112
5.  Tuning Riboswitch Regulation through Conformational Selection 
Journal of molecular biology  2010;405(4):926-938.
The SMK box riboswitch, which represents one of three known classes of S-adenosylmethionine (SAM)-responsive riboswitches, regulates gene expression in bacteria at the level of translation initiation. In contrast to most riboswitches, which contain separate domains responsible for ligand recognition and gene regulation, the ligand-binding and regulatory domains of the SMK box riboswitch are coincident. This property was exploited to allow the first atomic-level characterization of a functionally intact riboswitch in both the ligand-bound and ligand-free states. NMR spectroscopy revealed distinct mutually exclusive RNA conformations that are differentially populated in the presence or absence of the effector metabolite. Isothermal titration calorimetry and in vivo reporter assay results revealed the thermodynamic and functional consequences of this conformational equilibrium. We present a comprehensive model of the structural, thermodynamic, and functional properties of this compact RNA regulatory element.
PMCID: PMC3019289  PMID: 21075119
RNA; NMR spectroscopy; isothermal titration calorimetry; gene regulation; pre-existing equilibrium; riboswitch
6.  Assembly of the Complex between Archaeal RNase P Proteins RPP30 and Pop5 
Archaea  2011;2011:891531.
RNase P is a highly conserved ribonucleoprotein enzyme that represents a model complex for understanding macromolecular RNA-protein interactions. Archaeal RNase P consists of one RNA and up to five proteins (Pop5, RPP30, RPP21, RPP29, and RPP38/L7Ae). Four of these proteins function in pairs (Pop5-RPP30 and RPP21–RPP29). We have used nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC) to characterize the interaction between Pop5 and RPP30 from the hyperthermophilic archaeon Pyrococcus furiosus (Pfu). NMR backbone resonance assignments of free RPP30 (25 kDa) indicate that the protein is well structured in solution, with a secondary structure matching that observed in a closely related crystal structure. Chemical shift perturbations upon the addition of Pop5 (14 kDa) reveal its binding surface on RPP30. ITC experiments confirm a net 1 : 1 stoichiometry for this tight protein-protein interaction and exhibit complex isotherms, indicative of higher-order binding. Indeed, light scattering and size exclusion chromatography data reveal the complex to exist as a 78 kDa heterotetramer with two copies each of Pop5 and RPP30. These results will inform future efforts to elucidate the functional role of the Pop5-RPP30 complex in RNase P assembly and catalysis.
PMCID: PMC3227427  PMID: 22162665
7.  GUARDD: user-friendly MATLAB software for rigorous analysis of CPMG RD NMR data 
Journal of biomolecular NMR  2011;52(1):11-22.
Molecular dynamics are essential for life, and nuclear magnetic resonance (NMR) spectroscopy has been used extensively to characterize these phenomena since the 1950s. For the past 15 years, the Carr-Purcell Meiboom-Gill relaxation dispersion (CPMG RD) NMR experiment has afforded advanced NMR labs access to kinetic, thermodynamic, and structural details of protein and RNA dynamics in the crucial µs-ms time window. However, analysis of RD data is challenging because datasets are often large and require many non-linear fitting parameters, thereby confounding assessment of accuracy. Moreover, novice CPMG experimentalists face an additional barrier because current software options lack an intuitive user interface and extensive documentation. Hence, we present the open-source software package GUARDD (Graphical User-friendly Analysis of Relaxation Dispersion Data), which is designed to organize, automate, and enhance the analytical procedures which operate on CPMG RD data ( This MATLAB-based program includes a graphical user interface, permits global fitting to multi-field, multi-temperature, multi-coherence data, and implements χ2-mapping procedures, via grid-search and Monte Carlo methods, to enhance and assess fitting accuracy. The presentation features allow users to seamlessly traverse the large amount of results, and the RD Simulator feature can help design future experiments as well as serve as a teaching tool for those unfamiliar with RD phenomena. Based on these innovative features, we expect that GUARDD will fill a well-defined gap in service of the RD NMR community.
PMCID: PMC3593345  PMID: 22160811
Protein dynamics; RNA dynamics; Relaxation dispersion; Carr-Purcell Meiboom-Gill
8.  Structural basis for high-affinity binding of LEDGF PWWP to mononucleosomes 
Nucleic Acids Research  2013;41(6):3924-3936.
Lens epithelium-derived growth factor (LEDGF/p75) tethers lentiviral preintegration complexes (PICs) to chromatin and is essential for effective HIV-1 replication. LEDGF/p75 interactions with lentiviral integrases are well characterized, but the structural basis for how LEDGF/p75 engages chromatin is unknown. We demonstrate that cellular LEDGF/p75 is tightly bound to mononucleosomes (MNs). Our proteomic experiments indicate that this interaction is direct and not mediated by other cellular factors. We determined the solution structure of LEDGF PWWP and monitored binding to the histone H3 tail containing trimethylated Lys36 (H3K36me3) and DNA by NMR. Results reveal two distinct functional interfaces of LEDGF PWWP: a well-defined hydrophobic cavity, which selectively interacts with the H3K36me3 peptide and adjacent basic surface, which non-specifically binds DNA. LEDGF PWWP exhibits nanomolar binding affinity to purified native MNs, but displays markedly lower affinities for the isolated H3K36me3 peptide and DNA. Furthermore, we show that LEDGF PWWP preferentially and tightly binds to in vitro reconstituted MNs containing a tri-methyl-lysine analogue at position 36 of H3 and not to their unmodified counterparts. We conclude that cooperative binding of the hydrophobic cavity and basic surface to the cognate histone peptide and DNA wrapped in MNs is essential for high-affinity binding to chromatin.
PMCID: PMC3616739  PMID: 23396443
9.  Mechanisms of Allosteric Gene Regulation by NMR Quantification of μs-ms Protein Dynamics 
Journal of Molecular Biology  2011;415(2):372-381.
The trp RNA-binding attenuation protein (TRAP) is a paradigmatic allosteric protein that regulates the tryptophan biosynthetic genes associated with the trp operon in Bacilli. The ring-shaped 11-mer TRAP is activated for recognition of a specific trp-mRNA target by binding up to 11 tryptophan molecules. To characterize the mechanisms of tryptophan-induced TRAP activation we have performed methyl relaxation dispersion (MRD) nuclear magnetic resonance (NMR) experiments that probe the time-dependent structure of TRAP in the microsecond to millisecond “chemical exchange” time window. We find significant side chain flexibility localized to the RNA and tryptophan binding sites of the apo protein, and that these dynamics are dramatically reduced upon ligand binding. Analysis of the MRD NMR data provides insights into the structural nature of transiently populated conformations sampled in solution by apo TRAP. The MRD data are inconsistent with global two-state exchange, indicating that conformational sampling in apo TRAP is asynchronous. These findings imply a temporally heterogeneous population of structures that are incompatible with RNA binding, and substantiate the study of TRAP as a paradigm for probing and understanding essential dynamics in allosteric, regulatory proteins.
PMCID: PMC3258336  PMID: 22115774
TRAP; tryptophan; trp; CPMG; methyl relaxation dispersion
10.  Cooperative RNP assembly: Complementary rescue of structural defects by protein and RNA subunits of archaeal RNase P 
Journal of molecular biology  2011;411(2):368-383.
RNase P is a ribonucleoprotein (RNP) complex that utilizes a Mg2+-dependent RNA catalyst to cleave the 5′-leader of precursor tRNAs (pre-tRNAs) and generate mature tRNAs. The bacterial RNase P protein (RPP) aids RNase P RNA (RPR) catalysis by promoting substrate binding, Mg2+ coordination, and product release. Archaeal RNase P comprises an RPR and at least four RPPs, which have eukaryal homologs and function as two binary complexes (POP5•RPP30 and RPP21•RPP29). In this study, we employed a previously characterized substrate-enzyme conjugate [pre-tRNATyr-Methanocaldococcus jannaschii (Mja) RPR] to investigate the functional role of a universally conserved uridine in a bulge-helix structure in archaeal RPRs. Deletion of this bulged uridine resulted in an 80-fold decrease in the self-cleavage rate of pre-tRNATyr-MjaΔU RPR compared to the wildtype, and this defect was partially ameliorated upon addition of either RPP pair. The catalytic defect in the archaeal mutant RPR mirrors that reported in a bacterial RPR and highlights a parallel in their active sites. Furthermore, an N-terminal deletion mutant of Pyrococcus furiosus (Pfu) RPP29 that is defective in assembling with its binary partner RPP21, as assessed by isothermal titration calorimetry and NMR spectroscopy, is functional when reconstituted with the cognate Pfu RPR. Collectively, these results indicate that archaeal RPPs are able to compensate for structural defects in their cognate RPR and vice-versa, and provide striking examples of the cooperative subunit interactions critical for driving archaeal RNase P towards its functional conformation. (236 words)
PMCID: PMC3143260  PMID: 21683084
pre-tRNA processing; in vitro reconstitution; mutational rescue
11.  An introduction to NMR-based approaches for measuring protein dynamics 
Biochimica et biophysica acta  2010;1814(8):942-968.
Proteins are inherently flexible at ambient temperature. At equilibrium, they are characterized by a set of conformations that undergo continuous exchange within a hierarchy of spatial and temporal scales ranging from nanometers to micrometers and femtoseconds to hours. Dynamic properties of proteins are essential for describing the structural bases of their biological functions including catalysis, binding, regulation and cellular structure. Nuclear magnetic resonance (NMR) spectroscopy represents a powerful technique for measuring these essential features of proteins. Here we provide an introduction to NMR-based approaches for studying protein dynamics, highlighting eight distinct methods with recent examples, contextualized within a common experimental and analytical framework. The selected methods are (1) Real-time NMR, (2) Exchange spectroscopy, (3) Lineshape analysis, (4) CPMG relaxation dispersion, (5) Rotating frame relaxation dispersion, (6) Nuclear spin relaxation, (7) Residual dipolar coupling, (8) Paramagnetic relaxation enhancement.
PMCID: PMC3061256  PMID: 21059410
protein dynamics; protein flexibility; NMR
12.  Fidelity of tRNA 5′-maturation: a possible basis for the functional dependence of archaeal and eukaryal RNase P on multiple protein cofactors 
Nucleic Acids Research  2012;40(10):4666-4680.
RNase P, which catalyzes tRNA 5′-maturation, typically comprises a catalytic RNase P RNA (RPR) and a varying number of RNase P proteins (RPPs): 1 in bacteria, at least 4 in archaea and 9 in eukarya. The four archaeal RPPs have eukaryotic homologs and function as heterodimers (POP5•RPP30 and RPP21•RPP29). By studying the archaeal Methanocaldococcus jannaschii RPR's cis cleavage of precursor tRNAGln (pre-tRNAGln), which lacks certain consensus structures/sequences needed for substrate recognition, we demonstrate that RPP21•RPP29 and POP5•RPP30 can rescue the RPR's mis-cleavage tendency independently by 4-fold and together by 25-fold, suggesting that they operate by distinct mechanisms. This synergistic and preferential shift toward correct cleavage results from the ability of archaeal RPPs to selectively increase the RPR's apparent rate of correct cleavage by 11 140-fold, compared to only 480-fold for mis-cleavage. Moreover, POP5•RPP30, like the bacterial RPP, helps normalize the RPR's rates of cleavage of non-consensus and consensus pre-tRNAs. We also show that archaeal and eukaryal RNase P, compared to their bacterial relatives, exhibit higher fidelity of 5′-maturation of pre-tRNAGln and some of its mutant derivatives. Our results suggest that protein-rich RNase P variants might have evolved to support flexibility in substrate recognition while catalyzing efficient, high-fidelity 5′-processing.
PMCID: PMC3378863  PMID: 22298511
13.  Solution Structure of an Archaeal RNase P Binary Protein Complex. Formation of the 30-kDa Complex Between Pyrococcus furiosus RPP21 and RPP29 is Accompanied by Coupled Protein Folding, and Highlights Critical Features for Protein-Protein and Protein-RNA Interactions 
Journal of molecular biology  2009;393(5):1043-1055.
RNase P is a ribonucleoprotein (RNP) enzyme that catalyzes the Mg2+-dependent 5’ maturation of precursor tRNAs. In all domains of life, it is a ribozyme: the RNase P RNA (RPR) component has been demonstrated to be responsible for catalysis. However, the number of RNase P protein subunits (RPPs) varies from one in bacteria to nine or ten in eukarya. The archaeal RPR is associated with at least four RPPs, which function in pairs (RPP21–RPP29 and RPP30-POP5). We used solution NMR spectroscopy to determine the three-dimensional structure of the protein-protein complex comprising Pyrococcus furiosus (Pfu) RPP21 and RPP29. We found that the protein-protein interaction is characterized by coupled folding of secondary structural elements that participate in interface formation. In addition to detailing the intermolecular contacts that stabilize this 30-kDa binary complex, the structure identifies surfaces rich in conserved basic residues likely vital for recognition of the RPR and/or precursor tRNA. Furthermore, enzymatic footprinting experiments allowed us to localize the RPP21–RPP29 complex to the specificity domain of the RPR. These findings provide valuable new insights into mechanisms of RNP assembly and serve as important steps towards a three-dimensional model of this ancient RNP enzyme.
PMCID: PMC2782587  PMID: 19733182
14.  Ligand-induced changes in the structure and dynamics of E. coli peptide deformylase 
Biochemistry  2009;48(32):7595-7607.
Peptide deformylase (PDF) is an enzyme that is responsible for removing the formyl group from nascently synthesized polypeptides in bacteria, attracting much attention as a potential target for novel antibacterial agents. Efforts to develop potent inhibitors of the enzyme have progressed based on classical medicinal chemistry, combinatorial chemistry, and structural approaches. Yet, the validity of PDF as an antibacterial target hangs, in part, on the ability of inhibitors to selectively target this enzyme in favor of structurally related metallohydrolases. We have used 15N NMR spectroscopy and isothermal titration calorimetry to investigate the high-affinity interaction of EcPDF with actinonin, a naturally occurring potent EcPDF inhibitor. Backbone amide chemical shifts, residual dipolar couplings, hydrogen-deuterium exchange, and 15N relaxation reveal structural and dynamic effects of ligand binding in the immediate vicinity of the ligand binding site as well as at remote sites. A comparison of the crystal structures of free and actinonin-bound EcPDF with the solution data suggests that most of the consequences of ligand binding on the protein are lost or obscured during crystallization. The results of these studies improve our understanding of the thermodynamic global minimum and have important implications for structure-based drug design.
PMCID: PMC2881472  PMID: 19627112
15.  Crystallization and structure determination of the core-binding domain of bacteriophage lambda integrase 
The crystal structure of the core-binding domain of bacteriophage lambda integrase has been determined at 2.0 Å resolution.
Bacteriophage lambda integrase catalyzes site-specific DNA recombination. A helical bundle domain in the enzyme, called the core-binding domain (IntCB), promotes the catalysis of an intermediate DNA-cleavage reaction that is critical for recombination and is not well folded in solution in the absence of DNA. To gain structural insights into the mechanism behind the accessory role of this domain in catalysis, an attempt was made to crystallize an IntCB–DNA complex, but crystals of free IntCB were fortuitously obtained. The three-dimensional structure of DNA-free IntCB was solved at 2.0 Å resolution by molecular replacement using as the search model the previously available DNA-bound 2.8 Å structure of the IntCB domain in a larger construct of lambda integrase. The crystal structure of DNA-free IntCB resembles the DNA-bound structure of IntCB, but exhibits subtle differences in the DNA-binding face and lacks electron density for ten residues in the C-terminus that form a portion of a linker connecting IntCB to the C-terminal catalytic domain of the enzyme. Thus, this work reveals the domain in the absence of DNA and allows comparison with the DNA-bound form of this catalytically activating domain.
PMCID: PMC2496856  PMID: 18540053
bacteriophage lambda integrase; binding-coupled folding
16.  Solution NMR of large molecules and assemblies† 
Biochemistry  2007;46(2):331-340.
Solution NMR spectroscopy represents a powerful tool for examining the structure and function of biological macromolecules. The advent of multidimensional (2D–4D) NMR, together with the widespread use of uniform isotopic labeling of proteins and RNA with the NMR-active isotopes, 15N and 13C, opened the door to detailed analyses of macromolecular structure, dynamics and interactions of smaller macromolecules (< ~25 kDa). Over the past 10 years, advances in NMR and isotope labeling methods have expanded the range of NMR-tractable targets by at least an order of magnitude. Here we briefly describe the methodological advances that allow NMR spectroscopy of large macromolecules and their complexes, and provide a perspective on the wide range of applications of NMR to biochemical problems.
PMCID: PMC2596980  PMID: 17209543
17.  Thermodynamics of Tryptophan-Mediated Activation of the trp RNA-Binding Attenuation Protein (TRAP) 
Biochemistry  2006;45(25):7844-7853.
The trp RNA-binding attenuation protein (TRAP) functions in many Bacilli to control the expression of the tryptophan biosynthesis genes. Transcription of the trp operon is controlled by TRAP through an attenuation mechanism, in which competition between two alternative secondary structural elements in the 5′ leader sequence of the nascent mRNA is influenced by tryptophan-dependent binding of TRAP to the RNA. Previously, NMR studies of the undecamer (11-mer) suggested that tryptophan-dependent control of RNA binding by TRAP is accomplished through ligand-induced changes in protein dynamics. We now present further insights into this ligand-coupled event from hydrogen/deuterium (H/D) exchange analysis, differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC). Scanning calorimetry showed tryptophan dissociation to be independent of global protein unfolding, while analysis of the temperature dependence of the binding enthalpy by ITC revealed a negative heat capacity change larger than expected from surface burial, a hallmark of binding-coupled processes. Analysis of this excess heat capacity change using parameters derived from protein folding studies, corresponds to the ordering of 17-24 residues per monomer of TRAP upon tryptophan binding. This result is in agreement with qualitative analysis of residue-specific broadening observed in TROSY NMR spectra of the 91 kDa oligomer. Implications for the mechanism of ligand-mediated TRAP activation through a shift in a preexisting conformational equilibrium and an induced fit conformational change are discussed.
PMCID: PMC2517159  PMID: 16784236
calorimetry; binding-coupled protein folding; allosteric regulation; oligomer; trp RNA-binding attenuation protein; induced fit; pre-existing conformational equilibrium
18.  Trans cooperativity by a split DNA recombinase: the central and catalytic domains of bacteriophage lambda integrase cooperate in cleaving DNA substrates when the two domains are not covalently linked 
Journal of molecular biology  2007;370(2):303-314.
Site-specific recombinases of the λ-integrase family recognize and cleave their cognate DNA sites through cooperative binding to opposite sides of the DNA substrate by a C-terminal catalytic domain and a flexibly linked ‘core-binding’ domain; regulation of this cleavage is achieved via the formation of higher-order complexes. We report that the core-binding domain of λ-integrase is able to stimulate the activity of the catalytic domain even when the two domains are not linked. This trans stimulation is accomplished without significantly increasing the affinity of the catalytic domain for its DNA substrate. Moreover, we show that mutations in the DNA substrate can abrogate this effect while retaining specificity determinants for cleavage. Since the domains do not significantly interact directly, this finding implies that trans activation is achieved via the DNA substrate in a manner that may be mechanistically important in this and similar DNA binding and cleaving enzymes.
PMCID: PMC2034338  PMID: 17531268
lambda integrase; DNA recombinase; tyrosine recombinase; covalent intermediate; suicide substrate; substrate binding; DNA binding; NMR; single turnover kinetics; fluorescence anisotropy

Results 1-18 (18)