Structure of mitochondrial poly(A) RNA polymerase reveals the structural basis for dimerization, ATP selectivity and the SPAX4 disease phenotype.

Polyadenylation, performed by poly(A) polymerases (PAPs), is a ubiquitous post-transcriptional modification that plays key roles in multiple aspects of RNA metabolism. Although cytoplasmic and nuclear PAPs have been studied extensively, the mechanism by which mitochondrial PAP (mtPAP) selects adenosine triphosphate over other nucleotides is unknown. Furthermore, mtPAP is unique because it acts as a dimer.

Functional coupling of duplex translocation to DNA cleavage in a type I restriction enzyme.

Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA cleavage and ATP-dependent DNA translocation activities located on motor subunit HsdR. Functional coupling of DNA cleavage and translocation is a hallmark of the Type I restriction systems that is consistent with their proposed role in horizontal gene transfer. DNA cleavage occurs at nonspecific sites distant from the cognate recognition sequence, apparently triggered by stalled translocation.

Assembly Pathway and Characterization of the RAG1/2-DNA Paired and Signal-end Complexes.

Mammalian immune receptor diversity is established via a unique restricted set of site-specific DNA rearrangements in lymphoid cells, known as V(D)J recombination. The lymphoid-specific RAG1-RAG2 protein complex (RAG1/2) initiates this process by binding to two types of recombination signal sequences (RSS), 12RSS and 23RSS, and cleaving at the boundaries of RSS and V, D, or J gene segments, which are to be assembled into immunoglobulins and T-cell receptors. Here we dissect the ordered assembly of the RAG1/2 heterotetramer with 12RSS and 23RSS DNAs.

Crystal structure of the V(D)J recombinase RAG1-RAG2.

V(D)J recombination in the vertebrate immune system generates a highly diverse population of immunoglobulins and T-cell receptors by combinatorial joining of segments of coding DNA. The RAG1-RAG2 protein complex initiates this site-specific recombination by cutting DNA at specific sites flanking the coding segments. Here we report the crystal structure of the mouse RAG1-RAG2 complex at 3.

1.2 Å resolution crystal structure of Escherichia coli WrbA holoprotein.

The Escherichia coli protein WrbA, an FMN-dependent NAD(P)H:quinone oxidoreductase, was crystallized under new conditions in the presence of FAD or the native cofactor FMN. Slow-growing deep yellow crystals formed with FAD display the tetragonal bipyramidal shape typical for WrbA and diffract to 1.2 Å resolution, the highest yet reported.

Examining the role of the HIV-1 reverse transcriptase p51 subunit in positioning and hydrolysis of RNA/DNA hybrids.

Recent crystallographic analysis of p66/p51 human immunodeficiency virus (HIV) type 1 reverse transcriptase (RT) complexed with a non-polypurine tract RNA/DNA hybrid has illuminated novel and important contacts between structural elements at the C terminus of the noncatalytic p51 subunit and the nucleic acid duplex in the vicinity of the ribonuclease H (RNase H) active site. In particular, a short peptide spanning residues Phe-416-Pro-421 was shown to interact with the DNA strand, cross the minor groove of the helix, and then form Van der Waals contacts with the RNA strand adjacent to the scissile phosphate. At the base of the adjoining α-helix M', Tyr-427 forms a hydrogen bond with Asn-348, the latter of which, when mutated to Ile, is implicated in resistance to both nucleoside and non-nucleoside RT inhibitors.

Complexes of HIV-1 RT, NNRTI and RNA/DNA hybrid reveal a structure compatible with RNA degradation.

Hundreds of structures of type 1 human immunodeficiency virus (HIV-1) reverse transcriptase (RT) have been determined, but only one contains an RNA/DNA hybrid. Here we report three structures of HIV-1 RT complexed with a non-nucleotide RT inhibitor (NNRTI) and an RNA/DNA hybrid. In the presence of an NNRTI, the RNA/DNA structure differs from all prior nucleic acid-RT structures including the RNA/DNA hybrid.

Raman spectroscopy adds complementary detail to the high-resolution x-ray crystal structure of photosynthetic PsbP from Spinacia oleracea.

Raman microscopy permits structural analysis of protein crystals in situ in hanging drops, allowing for comparison with Raman measurements in solution. Nevertheless, the two methods sometimes reveal subtle differences in structure that are often ascribed to the water layer surrounding the protein. The novel method of drop-coating deposition Raman spectropscopy (DCDR) exploits an intermediate phase that, although nominally "dry," has been shown to preserve protein structural features present in solution.

Assembly of prototype foamy virus strand transfer complexes on product DNA bypassing catalysis of integration.

Integrase is the key enzyme that mediates integration of retroviral DNA into cellular DNA which is essential for viral replication. Inhibitors of HIV-1 that target integrase recognize the nucleoprotein complexes formed by integrase and viral DNA substrate (intasomes) rather than the free enzyme. Atomic resolution structures of HIV-1 intasomes are therefore required to understand the mechanisms of inhibition and drug resistance.

Structure of the motor subunit of type I restriction-modification complex EcoR124I.

Type I restriction-modification enzymes act as conventional adenine methylases on hemimethylated DNAs, but unmethylated recognition targets induce them to translocate thousands of base pairs before cleaving distant sites nonspecifically. The first crystal structure of a type I motor subunit responsible for translocation and cleavage suggests how the pentameric translocating complex is assembled and provides a structural framework for translocation of duplex DNA by RecA-like ATPase motors.

Crystals of DhaA mutants from Rhodococcus rhodochrous NCIMB 13064 diffracted to ultrahigh resolution: crystallization and preliminary diffraction analysis.

The enzyme DhaA from Rhodococcus rhodochrous NCIMB 13064 belongs to the haloalkane dehalogenases, which catalyze the hydrolysis of haloalkanes to the corresponding alcohols. The haloalkane dehalogenase DhaA and its variants can be used to detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Three mutants named DhaA04, DhaA14 and DhaA15 were constructed in order to study the importance of tunnels connecting the buried active site with the surrounding solvent to the enzymatic activity.

Purification, crystallization and preliminary X-ray analysis of the HsdR subunit of the EcoR124I endonuclease from Escherichia coli.

EcoR124I is a multicomplex enzyme belonging to the type I restriction-modification system from Escherichia coli. Although EcoR124I has been extensively characterized biochemically, there is no direct structural information available about particular subunits. HsdR is a motor subunit that is responsible for ATP hydrolysis, DNA translocation and cleavage of the DNA substrate recognized by the complex.