The positioning of nucleosomes along chromatin has been implicated in the regulation of gene expression in eukaryotic cells, because packaging DNA into nucleosomes affects sequence accessibility. We developed a tiled microarray approach to identify at high resolution the translational positions of 2278 nucleosomes over 482 kilobases of Saccharomyces cerevisiae DNA, including almost all of chromosome III and 223 additional regulatory regions. The majority of the nucleosomes identified were well-positioned. We found a stereotyped chromatin organization at Pol II promoters consisting of a nucleosome-free region approximately 200 base pairs upstream of the start codon flanked on both sides by positioned nucleosomes. The nucleosome-free sequences were evolutionarily conserved and were enriched in poly-deoxyadenosine or poly-deoxythymidine sequences. Most occupied transcription factor binding motifs were devoid of nucleosomes, strongly suggesting that nucleosome positioning is a global determinant of transcription factor access.

Chromatin plays roles in processes governed by different time scales. To assay the dynamic behavior of chromatin in living cells, we used genomic tiling arrays to measure histone H3 turnover in G1-arrested Saccharomyces cerevisiae at single-nucleosome resolution over 4% of the genome, and at lower (approximately 265 base pair) resolution over the entire genome. We find that nucleosomes at promoters are replaced more rapidly than at coding regions and that replacement rates over coding regions correlate with polymerase density. In addition, rapid histone turnover is found at known chromatin boundary elements. These results suggest that rapid histone turnover serves to functionally separate chromatin domains and prevent spread of histone states.

Summary Heat causes protein misfolding and aggregation, and in eukaryotic cells triggers aggregation of proteins and RNA into stress granules. We have carried out extensive proteomic studies to quantify heat-triggered aggregation and subsequent disaggregation in budding yeast, identifying more than 170 endogenous proteins aggregating within minutes of heat shock in multiple subcellular compartments. We demonstrate that these aggregated proteins are not misfolded and destined for degradation. Stable-isotope labeling reveals that even severely aggregated endogenous proteins are disaggregated without degradation during recovery from shock, contrasting with the rapid degradation observed for exogenous thermolabile proteins. Although aggregation likely inactivates many cellular proteins, in the case of a heterotrimeric aminoacyl-tRNA synthetase complex, the aggregated proteins remain active with unaltered fidelity. We propose that most heat-induced aggregation of mature proteins reflects the operation of an adaptive, autoregulatory process of functionally significant aggregate assembly and disassembly that aids cellular adaptation to thermal stress.

The histone code hypothesis holds that covalent posttranslational modifications of histone tails are interpreted by the cell to yield a rich combinatorial transcriptional output. This hypothesis has been the subject of active debate in the literature. Here, we investigated the combinatorial complexity of the acetylation code at the four lysine residues of the histone H4 tail in budding yeast. We constructed yeast strains carrying all 15 possible combinations of mutations among lysines 5, 8, 12, and 16 to arginine in the histone H4 tail, mimicking positively charged, unacetylated lysine states, and characterized the resulting genome-wide changes in gene expression by using DNA microarrays. Only the lysine 16 mutation had specific transcriptional consequences independent of the mutational state of the other lysines (affecting Ϸ100 genes). In contrast, for lysines 5, 8, and 12, expression changes were due to nonspecific, cumulative effects seen as increased transcription correlating with an increase in the total number of mutations (affecting Ϸ1,200 genes). Thus, acetylation of histone H4 is interpreted by two mechanisms: a specific mechanism for lysine 16 and a nonspecific, cumulative mechanism for lysines 5, 8, and 12.chromatin ͉ gene expression ͉ histone code ͉ nucleosomes

Evolving lineages face a constant intracellular threat: most new coding sequence mutations destabilize the folding of the encoded protein. Misfolded proteins form insoluble aggregates and are hypothesized to be intrinsically cytotoxic. Here, we experimentally isolate a fitness cost caused by toxicity of misfolded proteins. We exclude other costs of protein misfolding, such as loss of functional protein or attenuation of growth-limiting protein synthesis resources, by comparing growth rates of budding yeast expressing folded or misfolded variants of a gratuitous protein, YFP, at equal levels. We quantify a fitness cost that increases with misfolded protein abundance, up to as much as a 3.2% growth rate reduction when misfolded YFP represents less than 0.1% of total cellular protein.Comparable experiments on variants of the yeast gene orotidine-5′-phosphate decarboxylase (URA3) produce similar results. Quantitative proteomic measurements reveal that, within the cell, misfolded YFP induces coordinated synthesis of interacting cytosolic chaperone proteins in the absence of a wider stress response, providing evidence for an evolved modular response to misfolded proteins in the cytosol. These results underscore the distinct and evolutionarily relevant molecular threat of protein misfolding, independent of protein function. Assuming that most misfolded proteins impose similar costs, yeast cells express almost all proteins at steady-state levels sufficient to expose their encoding genes to selection against misfolding, lending credibility to the recent suggestion that such selection imposes a global constraint on molecular evolution.proteomics | stability | heat shock | evolutionary rate M ost new genetic mutations arising in protein-coding sequences decrease the likelihood that the encoding protein will fold properly (1, 2). Misfolding reduces the concentration of functional proteins, squanders cellular time and energy on production of useless proteins (3), and generates misfolded proteins that may harm cells (4). Given the high probability and diverse effects of failed protein folding, isolating and quantifying the influences of misfolding on fitness are essential for the development of mechanistic answers to fundamental questions in evolutionary biology: the distribution of fitness effects of new mutations, the interpretation of varying rates of molecular evolution, and the significance of compensatory mutations. Understanding how misfolding affects cell fitness may also illuminate the molecular basis of human diseases, particularly neurological disorders linked to misfolded protein toxicity (4, 5).Patterns of coding sequence evolution across taxa depend strongly on gene expression, with apparently limited contribution from protein function (6, 7). This has led to the misfolding hypothesis: that within-genome variation in purifying selection on coding sequences is predominantly shaped by the fitness cost of misfolded proteins and therefore, correlates with gene expression and protein abundance (8,9). It predicts that...

Introductory Paragraph Rod-shaped bacteria grow by adding material into their cell wall via the action of two spatially distinct enzymatic systems: The Rod complex moves around the cell circumference, while class A penicillin-binding proteins do not. To understand how the combined action of these two systems defines bacterial dimensions, we examined how each affects the growth and width of Bacillus subtilis, as well as the mechanical anisotropy and orientation of material within their sacculi. Rod width is not determined by MreB, rather it depends on the balance between the systems: The Rod complex reduces diameter, while aPBPs increase it. Increased Rod complex activity correlates with an increased density of directional MreB filaments and a greater fraction of directional PBP2a enzymes. This increased circumferential synthesis increases the relative amount of oriented material within the sacculi, making them more resistant to stretching across their width, thereby reinforcing rod shape. Together, these experiments explain how the combined action of the two main cell wall synthetic systems builds and maintains rods of different widths. Escherichia coli Rod mutants also show the same correlation between width and directional MreB filament density, suggesting this model may be generalizable to bacteria that elongate via the Rod complex.

20Rod shaped bacteria grow by adding material into their cell wall via the action of two spatially distinct enzymatic systems: 21The Rod system moves around the cell circumference, while the class A penicillin-binding proteins (aPBPs) are 22 unorganized. To understand how the combined action of these two systems defines bacterial dimensions, we examined how 23 each system affects the growth and width of Bacillus subtilis, as well as the mechanical anisotropy and orientation of 24 material within their sacculi. We find that rod diameter is not determined by MreB, rather it depends on the balance between 25 the systems: The Rod system reduces diameter, while aPBPs increase it. RodA/PBP2A can both thin or widen cells, 26 depending on its levels relative to MreBCD. Increased Rod system activity correlates with an increased density of directional 27MreB filaments, and a greater fraction of directionally moving PBP2A molecules. This increased circumferential synthesis 28 increases the amount of oriented material within the sacculi, increasing their mechanical anisotropy and reinforcing rod 29 shape. Together, these experiments explain how the combined action of the two main cell wall synthetic systems build rods 30 of different widths, a model that appears generalizable: Escherichia coli containing Rod system mutants show the same 31 relationship between the density of directionally moving MreB filaments and cell width.

Summary Neuromodulatory cells transduce environmental information into long lasting behavioral responses. However, the mechanisms governing how neuronal cells influence behavioral plasticity are difficult to characterize. Here, we adapted the Translating Ribosome Affinity Purification (TRAP) approach in C. elegans to profile ribosome-associated mRNAs from three major tissues and the neuromodulatory dopaminergic and serotonergic cells. We identified elc-2, an Elongin C ortholog, specifically expressed in stress-sensing ADF serotonergic sensory neurons, and found that it plays a role in mediating a long-lasting change in serotonin-dependent feeding behavior induced by heat stress. We demonstrate that ELC-2 and the von Hippel-Lindau protein VHL-1, components of an Elongin-CullinSOCS-box (ECS) E3 ubiquitin ligase, modulate this behavior after experiencing stress. Also, heat stress induces a transient redistribution of ELC-2, becoming more nuclearly enriched. Together, our results demonstrate dynamic regulation of an E3 ligase, and a role for an ECS complex in neuromodulation and control of lasting behavioral states.