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The survival of motor neurons (SMN) protein, product of the disease gene of the common neurodegenerative disease spinal muscular atrophy, is part of the large multiprotein "SMN complex." The SMN complex functions as an assembly machine for small nuclear ribonucleoproteins (snRNPs)-the major components of the spliceosome. Here, we report the crystal structure of two components of the human SMN complex, Gemin6 and Gemin7. Although Gemin6 and Gemin7 have no significant sequence similarity with Sm proteins, both adopt canonical Sm folds. Moreover, Gemin6 and Gemin7 exist as a heterodimer, and interact with each other via an interface similar to that which mediates interactions among the Sm proteins. Together with binding experiments that show that the Gemin6/Gemin7 complex binds to Sm proteins, these findings provide a framework for considering how the SMN complex, with Gemin6 and Gemin7 as tools, might organize Sm proteins for formation of Sm rings on snRNA targets.

Cre recombinase is a prototypical member of the tyrosine recombinase family of site-specific recombinases. Members of this family of enzymes catalyze recombination between specific DNA sequences by cleaving and exchanging one pair of strands between the two substrate sites to form a 4-way Holliday junction (HJ) intermediate and then resolve the HJ intermediate to recombinant products by a second round of strand exchanges. Recently, hexapeptide inhibitors have been described that are capable of blocking the second strand exchange step in the tyrosine recombinase recombination pathway, leading to an accumulation of the HJ intermediate. These peptides are active in the lambda-integrase, Cre recombinase, and Flp recombinase systems and are potentially important tools for both in vitro mechanistic studies and as in vivo probes of cellular function. Here we present biochemical and crystallographic data that support a model where the peptide inhibitor binds in the center of the recombinase-bound DNA junction and interacts with solvent-exposed bases near the junction branch point. Peptide binding induces large conformational changes in the DNA strands of the HJ intermediate, which affect the active site geometries in the recombinase subunits.

Background: Splicing of pre-mRNA in eukaryotes imprints the resulting mRNA with a specific multiprotein complex, the exon-exon junction complex (EJC), at the sites of intron removal. The proteins of the EJC, Y14, Magoh, Aly/REF, RNPS1, Srm160, and Upf3, play critical roles in postsplicing processing, including nuclear export and cytoplasmic localization of the mRNA, and the nonsense-mediated mRNA decay (NMD) surveillance process. Y14 and Magoh are of particular interest because they remain associated with the mRNA in the same position after its export to the cytoplasm and require translation of the mRNA for removal. This tenacious, persistent, splicing-dependent, yet RNA sequence-independent, association suggests an important signaling function and must require distinct structural features for these proteins., Results: We describe the high-resolution structure and biochemical properties of the highly conserved human Y14 and Magoh proteins. Magoh has an unusual structure comprised of an extremely flat, six-stranded anti-parallel beta sheet packed against two helices. Surprisingly, Magoh binds with high affinity to the RNP motif RNA binding domain (RBD) of Y14 and completely masks its RNA binding surface., Conclusions: The structure and properties of the Y14-Magoh complex suggest how the pre-mRNA splicing machinery might control the formation of a stable EJC-mRNA complex at splice junctions.

Cre recombinase is now widely used to carry out complex manipulations of DNA molecules both in vitro and in vivo. For in vitro experiments, there is a clear need for highly pure preparations of Cre and of Cre mutants that serve as controls or supply an altered activity or specificity. In vivo experiments utilizing Cre variants also often require in vitro characterization and some applications involve transfection of purified enzyme to achieve transient activity in the cell. This review outlines a detailed protocol for purification of native Cre and describes straightforward assays that can be used to test for recombination activity in vitro. The design of experiments to trap the intermediates of Cre-loxP site-specific recombination for biophysical studies is also presented. The methods described should be useful to any investigator with a need for purified Cre recombinase and should be broadly applicable to related site-specific recombination systems.

Structural models of site-specific recombinases from the lambda integrase family of enzymes have in the last four years provided an important new perspective on the three-dimensional nature of the recombination pathway. Members of this family, which include the bacteriophage P1 Cre recombinase, bacteriophage lambda integrase, the yeast Flp recombinase, and the bacterial XerCD recombinases, exchange strands between DNA substrates in a stepwise process. One pair of strands is exchanged to form a Holliday junction intermediate, and the second pair of strands is exchanged during resolution of the junction to products. Crystal structures of reaction intermediates in the Cre-loxP site-specific recombination system, together with recent biochemical studies in the field, support a "strand swapping" model for recombination that does not require branch migration of the Holliday junction intermediate in order to test homology between recombining sites.

The hexameric regulatory protein ArgR formed by arginine-mediated dimerization of identical trimers governs the expression of genes required for arginine metabolism and some other genes in mesophilic and moderately thermophilic bacteria. We have cloned the argR gene from two hyperthermophilic bacteria of the genus Thermotoga. The two-domain ArgR proteins encoded by T. neapolitana and T. maritima share a low degree of sequence similarity with other bacterial arginine repressors. The ArgR protein from T. neapolitana binds to an operator located just upstream of its coding sequence and, therefore, the argR gene may be autoregulated. The protein has extremely high intrinsic thermostability and tolerance to urea. Moreover, its binding to target DNA increases the melting temperature by approximately 15 degrees C. The formation of oligomeric ArgR-DNA complexes is a function of protein concentration, with hexameric complexes being favoured at higher concentrations. In the presence of arginine the hyperthermophilic ArgR protein binds to its own operator, argRo, only by forming hexamer ArgR-DNA complexes, whereas both trimer-DNA and hexamer-DNA complexes are detected in the absence of arginine. However, the affinity of T. neapolitana ArgR for DNA has been found to be higher for a mixture of trimers and non-bound hexamers than for arginine-bound hexamers. Our data indicate that genes for arginine biosynthesis are clustered in a putative operon, which could also be regulated by the ArgR protein, in the hyperthermophilic host.

Abstract Cre recombinase is a member of a large family of site-specific recombination enzymes that performs a cut-and-paste operation between two specific DNA sequences. Our goal has been to understand the mechanism of this complex reaction by trapping and characterizing the three-dimensional structures of each of the reaction intermediates. This work has led to high resolution crystallographic models of (i) the initial synaptic complex, (ii) the covalent Cre- DNA intermediate, and (iii) the Holliday junction intermediate. The Cre-loxP system appears to function by creating at the outset a protein-DNA architecture that resembles that of the Holliday junction intermediate that is eventually formed. The "arms" of the loxP sites are initially bent by about 75° in the synaptic complex, forming a nearly planar arrangement that is held fixed, while cleavage and strand exchange occur in the central region between the arms. The simplest view of the recombination pathway is that it contains two symmetrical halves, each of which uses this Holliday junction-like architectural framework to mediate the cleavage and ligation steps. The two halves are linked by a subtle isomerization of the Holliday intermediate that switches the roles of the recombinase subunits and allows exchange of the second pair of DNA strands. In this paper, we summarize recent structural results from our laboratory, with an emphasis on the geometry of the DNA substrates.

Cre recombinase catalyzes site-specific recombination between two 34-bp loxP sites in a variety of DNA substrates. At the start of the recombination pathway, the loxP sites are each bound by two recombinase molecules, and synapsis of the sites is mediated by Cre-Cre interactions. We describe the structures of synaptic complexes formed between a symmetrized loxP site and two Cre mutants that are defective in strand cleavage. The DNA in these complexes is bent sharply at a single base pair step at one end of the crossover region in a manner that is atypical of protein-induced DNA bends. A large negative roll (-49 degrees) and a positive tilt (16 degrees) open the major groove toward the center of the synapse and compress the minor groove toward the protein-DNA interface. The bend direction of the site appears to determine which of the two DNA substrate strands will be cleaved and exchanged in the initial stages of the recombination pathway. These results provide a structural basis for the observation that exchange of DNA strands proceeds in a defined order in some tyrosine recombinase systems. The Cre-loxS synaptic complex structure supports a model in which synapsis of the loxP sites results in formation of a Holliday junction-like DNA architecture that is maintained through the initial cleavage and strand exchange steps in the site-specific recombination pathway.

The arginine repressor (ArgR) is a hexameric DNA-binding protein that plays a multifunctional role in the bacterial cell. Here, we present the 2.5 A structure of apo-ArgR from Bacillus stearothermophilus and the 2.2 A structure of the hexameric ArgR oligomerization domain with bound arginine. This first view of intact ArgR reveals an approximately 32-symmetric hexamer of identical subunits, with six DNA-binding domains surrounding a central oligomeric core. The difference in quaternary organization of subunits in the arginine-bound and apo forms provides a possible explanation for poor operator binding by apo-ArgR and for high affinity binding in the presence of arginine.

We have determined the X-ray crystal structures of two DNA Holliday junctions (HJs) bound by Cre recombinase. The HJ is a four-way branched structure that occurs as an intermediate in genetic recombination pathways, including site-specific recombination by the lambda-integrase family. Cre recombinase is an integrase family member that recombines 34 bp loxP sites in the absence of accessory proteins or auxiliary DNA sequences. The 2.7 A structure of Cre recombinase bound to an immobile HJ and the 2.5 A structure of Cre recombinase bound to a symmetric, nicked HJ reveal a nearly planar, twofold-symmetric DNA intermediate that shares features with both the stacked-X and the square conformations of the HJ that exist in the unbound state. The structures support a protein-mediated crossover isomerization of the junction that acts as the switch responsible for activation and deactivation of recombinase active sites. In this model, a subtle isomerization of the Cre recombinase-HJ quaternary structure dictates which strands are cleaved during resolution of the junction via a mechanism that involves neither branch migration nor helical restacking.

Arginine biosynthesis in Escherichia coli is negatively regulated by the hexameric repressor protein ArgR and the corepressor L-arginine. L-Arginine binds to ArgR in the C-terminal domain of the repressor. Binding to operator DNA occurs in the N-terminal domain. The molecular structures of both domains have recently been elucidated. The known stereochemistry of the arginine binding pocket was used for the rational design of a mutant ArgR with altered ligand specificity. Our prediction was that a replacement of Asp128 by asparagine would preferentially lead to the binding of L-citrulline, rather than L-arginine. The D128N mutant was constructed and was shown to fulfill our expectation by several experimental approaches. By isothermal titration calorimetry it was found to bind L-citrulline much more strongly than L-arginine, in contrast to wild-type ArgR. Exchange between the mutant trimers of the hexamer was inhibited by L-citrulline, as it is by L-arginine in the wild-type. The mutant protein was precipitated by L-citrulline but not by L-arginine, whereas the reverse is true for the wild-type protein. Demonstration of a corepressor action was, however, precluded by the superrepressor effect of the D128N mutation by itself. The mutant protein, in the absence of L-citrulline or L-arginine is as strong a repressor as the wild-type protein in the presence of L-arginine. We discuss two possible mechanisms, in terms of the known domain structures that could explain our observations., (Copyright 1998 Academic Press Limited.)

During site-specific DNA recombination, which brings about genetic rearrangement in processes such as viral integration and excision and chromosomal segregation, recombinase enzymes recognize specific DNA sequences and catalyse the reciprocal exchange of DNA strands between these sites. The bacteriophage recombinase Cre catalyses site-specific recombination between two 34-base-pair loxP sites. The crystal structure at 2.4 A resolution of Cre bound to a loxP substrate reveals an intermediate in the recombination reaction, in which a Cre molecule has cleaved the substrate to form a covalent 3'-phosphotyrosine linkage with the DNA. Four recombinases and two loxP sites form a synapsed structure in which the DNA resembles models of four-way Holliday-Junction intermediates. The Cre-loxP complex challenges models of site-specific recombination that require large changes in quaternary structure. Subtle allosteric changes at the carboxy termini of the Cre subunits may instead coordinate the cleavage and strand-exchange reactions.

Four new studies of the assembly of different subsets of the preinitiation complex of RNA polymerase II have provided insight into how transcription factors interact with each other and with DNA. A heterotetramer of TBP-associated factors shows a histone-like fold.

The structure of the oligomerization and L-arginine binding domain of the Escherichia coli arginine repressor (ArgR) has been determined using X-ray diffraction methods at 2.2 A resolution with bound arginine and at 2.8 A in the unliganded form. The oligomeric core is a 3-fold rotationally symmetric hexamer formed from six identical subunits corresponding to the 77 C-terminal residues (80 to 156) of ArgR. Each subunit has an alpha/beta fold containing a four-stranded antiparallel beta-sheet and two antiparallel alpha-helices. The hexamer is formed from two trimers, each with tightly packed hydrophobic cores. In the absence of arginine, the trimers stack back-to-back through a dyad-symmetric, sparsely packed hydrophobic interface. Six molecules of arginine bind at the trimer-trimer interface, each making ten hydrogen bonds to the protein including a direct ion pair that crosslinks the two protein trimers. Solution experiments with wild-type ArgR and oligomerization domain indicate that the hexameric form is greatly stabilized upon arginine binding. The crystal structures and solution experiments together suggest possible mechanisms of how arginine activates ArgR to bind to its DNA targets and provides a stereochemical basis for interpreting the results of mutagenesis and biochemical experiments with ArgR.

High resolution structures for the complexes formed by the immunosuppressive agents FK506 and rapamycin with the human immunophilin FKBP-12 have been determined by X-ray diffraction. FKBP-12 has a novel fold comprised of a five-stranded beta-sheet wrapping around a short alpha-helix with an overall conical shape. Both FK506 and rapamycin bind in the cavity defined by the beta-sheet, alpha-helix and three loops. Both FK506 and rapamycin bind in similar fashions with a set of hydrogen bonds and an unusual carbonyl binding pocket. Bound FK506 has a different conformation than free (crystalline) FK506 while rapamycin's bound conformation is virtually identical to that of unbound rapamycin. FKBP-12 is a peptidyl-prolyl isomerase (PPIase), and the structures of the complexes suggest ways in which this catalytic activity could operate. The different complexes are active in suppressing different steps of T cell activation, an activity seemingly unconnected with the PPIase activity.

The structure of the human FK506 binding protein (FKBP), complexed with the immunosuppressant FK506, has been determined to 1.7 angstroms resolution by x-ray crystallography. The conformation of the protein changes little upon complexation, but the conformation of FK506 is markedly different in the bound and unbound forms. The drug's association with the protein involves five hydrogen bonds, a hydrophobic binding pocket lined with conserved aromatic residues, and an unusual carbonyl binding pocket. The nature of this complex has implications for the mechanism of rotamase catalysis and for the biological actions of FK506 and rapamycin.

Two sesquiterpenes have been isolated from the fungal pathogen of Bermuda grass Bipolaris cynodontis. Chemical, spectral, and x-ray diffraction studies have led to the characterization of these as bipolaroxin and dihydrobipolaroxin, highly oxygenated members of the eremophilane family. Bipolaroxin is phytotoxic to some but not all of the plants tested. To our knowledge, a phytotoxin with host selectivity isolated from a weed pathogen has not been reported previously.

Drechslera maydis, the causal agent of Southern corn leaf blight, and Drechslera sorghicola, the causal agent of leaf spot on Johnson grass, produce a series of phytotoxic sesterterpenoids. These sesterterpenoids belong to the ophiobolin family. One of them, ophiobolin I, was characterized by x-ray diffraction and served as a crucial reference compound for characterizing four other ophiobolins. All of the ophiobolins studied produce characteristic lesions on host plants at concentrations of 1 mM to 1 muM. The ophiobolin characterized as 6-epiophiobolin A is selectively toxic to corn bearing Texas-male-sterile (Tms) cytoplasm when assayed in a dark CO(2) fixation assay. It is plausible that these ophiobolins had a role in the 1970 corn-blight epidemic in North America.