The heptameric Nup84 complex constitutes an evolutionarily conserved building block of the nuclear pore complex. Here, we present the crystal structure of the heterotrimeric Sec13⅐Nup145C⅐Nup84 complex, the centerpiece of the heptamer, at 3.2-Å resolution. Nup84 forms a U-shaped ␣-helical solenoid domain, topologically similar to two other members of the heptamer, Nup145C and Nup85. The interaction between Nup84 and Nup145C is mediated via a hydrophobic interface located in the kink regions of the two solenoids that is reinforced by additional interactions of two long Nup84 loops. The Nup84 binding site partially overlaps with the homo-dimerization interface of Nup145C, suggesting competing binding events. Fitting of the elongated Z-shaped heterotrimer into electron microscopy (EM) envelopes of the heptamer indicates that structural changes occur at the Nup145C⅐Nup84 interface. Docking the crystal structures of all heptamer components into the EM envelope constitutes a major advance toward the completion of the structural characterization of the Nup84 complex.electron microscopy docking ͉ nuclear pore complex ͉ protein-protein interaction ͉ X-ray crystallography ͉ binding promiscuity

Rtt109 is a protein acetyltransferase (PAT) that is responsible for the acetylation of lysine-56 of histone 3 (H3K56) in yeast. H3K56 acetylation has been implicated in the weakening of the interaction between the histone core and the surrounding DNA in the nucleosomal particle. Rtt109, in cooperation with various histone chaperones, promotes genomic stability and is required for resistance to DNA damaging agents. Here, we present the crystal structure of Rtt109 in complex with acetyl-CoA at a 2.0-Å resolution. Rtt109 consists of a core PAT domain, which binds the acetyl-CoA cofactor. A second domain, the activation domain, is tightly associated with the PAT domain. Autoacetylation of lysine-290 within the activation domain is required for stabilizing the interaction between the two domains and is essential for catalysis. Biochemical analysis demonstrates the requirement of a loop within the PAT domain for the binding of the histone chaperone Vps75, and mutational analysis identifies key residues for catalysis. We propose a model in which the autoacetylation of Rtt109 is crucial for the regulation of its catalytic activity.DNA repair ͉ histone ͉ structure E ukaryotic genomic DNA is organized into a compact structure called chromatin. Chromatin formation is mainly established by a set of proteins termed histones. Histones are small globular proteins that are positively charged with flexible, N-terminal tails Ϸ40 residues in length. An octamer of histones, namely a H3/H4 tetramer that assembles with two H2A/H2B dimers, interacts with 147 bp of helical DNA to form the fundamental unit of chromatin, the nucleosomal particle (1). Histones undergo a variety of posttranslational modifications that are either attached or removed by enzyme families of counteracting activity (2, 3). The majority of the modifications are concentrated on the flexible N-terminal tails, although residues within the histone fold have also been found to be modified (4).Recently, the functionally important acetylation of lysine-56 of histone H3 (Ac-H3K56), located within the folded histone core, has been described (5-9). Structural studies have revealed that H3K56 contributes to the formation of the stable quaternary structure of the nucleosome. The acetylation of H3K56 neutralizes the positive charge of lysine-56, disrupting the electrostatic interaction between the helical DNA of the nucleosomal complex, potentially resulting in a more flexible wrapping of DNA around the proteinaceous histone core (1). Acetylation of H3K56 is predominately seen on newly synthesized histones during the S phase of the meiotic or mitotic cell cycle (6, 9, 10). Yeast mutants that cannot acetylate H3K56 because of the expression of an unmodifiable histone H3 variant in which arginine replaces lysine-56 (H3-K56R) are sensitive to genotoxic compounds during the S phase of the cell cycle and display an accumulation of stalled replication forks (6, 9-11). Moreover, the acetylation of H3K56 has been associated with the repair of DNA lesions that occur during replication...

Group II introns are self-splicing, retrotransposable ribozymes that contribute to gene expression and evolution in most organisms. The ongoing identification of new group II introns and recent bioinformatic analyses have suggested that there are novel lineages, which include the group IIE and IIF introns. Because the function and biochemical activity of group IIE and IIF introns have never been experimentally tested and because these introns appear to have features that distinguish them from other introns, we set out to determine if they were indeed self-splicing, catalytically active RNA molecules. To this end, we transcribed and studied a set of diverse group IIE and IIF introns, quantitatively characterizing their in vitro self-splicing reactivity, ionic requirements, and reaction products. In addition, we used mutational analysis to determine the relative role of the EBS-IBS 1 and 2 recognition elements during splicing by these introns. We show that group IIE and IIF introns are indeed distinct active intron families, with different reactivities and structures. We show that the group IIE introns self-splice exclusively through the hydrolytic pathway, while group IIF introns can also catalyze transesterifications. Intriguingly, we observe one group IIF intron that forms circular intron. Finally, despite an apparent EBS2-IBS2 duplex in the sequences of these introns, we find that this interaction plays no role during self-splicing in vitro. It is now clear that the group IIE and IIF introns are functional ribozymes, with distinctive properties that may be useful for biotechnological applications, and which may contribute to the biology of host organisms.

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