Architecture of the nuclear pore complex coat

The nuclear pore complex (NPC) constitutes the sole gateway for bidirectional nucleocytoplasmic transport. Despite half a century of structural characterization, the architecture of the NPC remains unknown. Here, we present the crystal structure of a reconstituted ~400 kDa coat nucleoporin complex (CNC) from S. cerevisiae at a 7.4-Å resolution. The crystal structure revealed a curved Y-shaped architecture and the molecular details of the coat nucleoporin interactions forming the central “triskelion” of the Y. A structural comparison of the yeast CNC with an electron microscopy reconstruction of its human counterpart suggested the evolutionary conservation of the elucidated architecture. Moreover, 32 copies of the CNC crystal structure docked readily into a cryoelectron tomographic reconstruction of the fully-assembled human NPC, thereby accounting for ~16 MDa of its mass

An Assessment of Dynamical Mass Constraints on Pre-Main Sequence Evolutionary Tracks

[abridged] We have assembled a database of stars having both masses determined from measured orbital dynamics and sufficient spectral and photometric information for their placement on a theoretical HR diagram. Our sample consists of 115 low mass (M < 2.0 Msun) stars, 27 pre-main sequence and 88 main sequence. We use a variety of available pre-main sequence evolutionary calculations to test the consistency of predicted stellar masses with dynamically determined masses. Despite substantial improvements in model physics over the past decade, large systematic discrepancies still exist between empirical and theoretically derived masses. For main-sequence stars, all models considered predict masses consistent with dynamical values above 1.2 Msun, some models predict consistent masses at solar or slightly lower masses, and no models predict consistent masses below 0.5 Msun but rather all models systematically under-predict such low masses by 5-20%. The failure at low masses stems from the poor match of most models to the empirical main-sequence below temperatures of 3800 K where molecules become the dominant source of opacity and convection is the dominant mode of energy transport. For the pre-main sequence sample we find similar trends. There is generally good agreement between predicted and dynamical masses above 1.2 Msun for all models. Below 1.2 Msun and down to 0.3 Msun (the lowest mass testable) most evolutionary models systematically under-predict the dynamically determined masses by 10-30% on average with the Lyon group models (e.g. Baraffe et al. 1998) predicting marginally consistent masses *in the mean* though with large scatter.Comment: accepted for publication in ApJ (2004

A Search for "Dwarf" Seyfert Nuclei. IV. Nuclei with Broad H-alpha Emission

We present the results of an optical spectroscopic survey designed to search for low-luminosity, "dwarf" Seyfert nuclei in a magnitude-limited sample of 486 bright, northern galaxies. Moderate-resolution spectra of exceptionally high quality were obtained in part to detect broad H-alpha emission, similar in character to, but much weaker than, the broad permitted lines that define type 1 Seyfert nuclei. One of the primary goals of the survey is to better quantify the faint end of the luminosity function of active galactic nuclei. This paper describes the subset of nuclei showing definite or probable evidence of broad H-alpha emission. We outline the procedures for determining the presence of this elusive spectral feature, steps for its quantitative measurement, and the associated systematic errors. Of the 211 emission-line galaxies classified as having Seyfert or LINER nuclei in our survey, the broad H-alpha line was detected with confidence in 34 objects, and with less certainty in another 12. Most of the detections are reported for the first time, and the detection rate represents a lower limit to the true incidence of active nuclei harboring a broad emission-line region. These statistics imply that broad-lined active nuclei are much more common than previously believed: they exist in at least 20% of all galaxies spectroscopically classified as "active" and in more than 10% of all luminous galaxies at the current epoch.Comment: To appear in the Astrophysical Journal Supplements. LaTex, 32 pages, plus an additional 14 figures and 3 tables. AASTex macro aaspp4.st

Pore timing:the evolutionary origins of the nucleus and nuclear pore complex

The name “eukaryote” is derived from Greek, meaning “true kernel”, and describes the domain of organisms whose cells have a nucleus. The nucleus is thus the defining feature of eukaryotes and distinguishes them from prokaryotes (Archaea and Bacteria), whose cells lack nuclei. Despite this, we discuss the intriguing possibility that organisms on the path from the first eukaryotic common ancestor to the last common ancestor of all eukaryotes did not possess a nucleus at all—at least not in a form we would recognize today—and that the nucleus in fact arrived relatively late in the evolution of eukaryotes. The clues to this alternative evolutionary path lie, most of all, in recent discoveries concerning the structure of the nuclear pore complex. We discuss the evidence for such a possibility and how this impacts our views of eukaryote origins and how eukaryotes have diversified subsequent to their last common ancestor

Mutant Versions of the S. cerevisiae Transcription Elongation Factor Spt16 Define Regions of Spt16 That Functionally Interact with Histone H3

In eukaryotic cells, the highly conserved FACT (FAcilitates Chromatin Transcription) complex plays important roles in several chromatin-based processes including transcription initiation and elongation. During transcription elongation, the FACT complex interacts directly with nucleosomes to facilitate histone removal upon RNA polymerase II (Pol II) passage and assists in the reconstitution of nucleosomes following Pol II passage. Although the contribution of the FACT complex to the process of transcription elongation has been well established, the mechanisms that govern interactions between FACT and chromatin still remain to be fully elucidated. Using the budding yeast Saccharomyces cerevisiae as a model system, we provide evidence that the middle domain of the FACT subunit Spt16 – the Spt16-M domain – is involved in functional interactions with histone H3. Our results show that the Spt16-M domain plays a role in the prevention of cryptic intragenic transcription during transcription elongation and also suggest that the Spt16-M domain has a function in regulating dissociation of Spt16 from chromatin at the end of the transcription process. We also provide evidence for a role for the extreme carboxy terminus of Spt16 in functional interactions with histone H3. Taken together, our studies point to previously undescribed roles for the Spt16 M-domain and extreme carboxy terminus in regulating interactions between Spt16 and chromatin during the process of transcription elongation

FACT Prevents the Accumulation of Free Histones Evicted from Transcribed Chromatin and a Subsequent Cell Cycle Delay in G1

The FACT complex participates in chromatin assembly and disassembly during transcription elongation. The yeast mutants affected in the SPT16 gene, which encodes one of the FACT subunits, alter the expression of G1 cyclins and exhibit defects in the G1/S transition. Here we show that the dysfunction of chromatin reassembly factors, like FACT or Spt6, down-regulates the expression of the gene encoding the cyclin that modulates the G1 length (CLN3) in START by specifically triggering the repression of its promoter. The G1 delay undergone by spt16 mutants is not mediated by the DNA–damage checkpoint, although the mutation of RAD53, which is otherwise involved in histone degradation, enhances the cell-cycle defects of spt16-197. We reveal how FACT dysfunction triggers an accumulation of free histones evicted from transcribed chromatin. This accumulation is enhanced in a rad53 background and leads to a delay in G1. Consistently, we show that the overexpression of histones in wild-type cells down-regulates CLN3 in START and causes a delay in G1. Our work shows that chromatin reassembly factors are essential players in controlling the free histones potentially released from transcribed chromatin and describes a new cell cycle phenomenon that allows cells to respond to excess histones before starting DNA replication

FACT, the Bur Kinase Pathway, and the Histone Co-Repressor HirC Have Overlapping Nucleosome-Related Roles in Yeast Transcription Elongation

Gene transcription is constrained by the nucleosomal nature of chromosomal DNA. This nucleosomal barrier is modulated by FACT, a conserved histone-binding heterodimer. FACT mediates transcription-linked nucleosome disassembly and also nucleosome reassembly in the wake of the RNA polymerase II transcription complex, and in this way maintains the repression of ‘cryptic’ promoters found within some genes. Here we focus on a novel mutant version of the yeast FACT subunit Spt16 that supplies essential Spt16 activities but impairs transcription-linked nucleosome reassembly in dominant fashion. This Spt16 mutant protein also has genetic effects that are recessive, which we used to show that certain Spt16 activities collaborate with histone acetylation and the activities of a Bur-kinase/Spt4–Spt5/Paf1C pathway that facilitate transcription elongation. These collaborating activities were opposed by the actions of Rpd3S, a histone deacetylase that restores a repressive chromatin environment in a transcription-linked manner. Spt16 activity paralleling that of HirC, a co-repressor of histone gene expression, was also found to be opposed by Rpd3S. Our findings suggest that Spt16, the Bur/Spt4–Spt5/Paf1C pathway, and normal histone abundance and/or stoichiometry, in mutually cooperative fashion, facilitate nucleosome disassembly during transcription elongation. The recessive nature of these effects of the mutant Spt16 protein on transcription-linked nucleosome disassembly, contrasted to its dominant negative effect on transcription-linked nucleosome reassembly, indicate that mutant FACT harbouring the mutant Spt16 protein competes poorly with normal FACT at the stage of transcription-linked nucleosome disassembly, but effectively with normal FACT for transcription-linked nucleosome reassembly. This functional difference is consistent with the idea that FACT association with the transcription elongation complex depends on nucleosome disassembly, and that the same FACT molecule that associates with an elongation complex through nucleosome disassembly is retained for reassembly of the same nucleosome