TOR-dependent reduction in the expression level of Rrn3p lowers the activity of the yeast RNA Pol I machinery, but does not account for the strong inhibition of rRNA production.

Ribosome biogenesis is tightly linked to cellular growth. A crucial step in the regulation of ribosomal RNA (rRNA) gene transcription is the formation of the complex between RNA polymerase I (Pol I) and the Pol I-dependent transcription factor Rrn3p. We found that TOR inactivation leads to proteasome-dependent degradation of Rrn3p and a strong reduction in initiation competent Pol I-Rrn3p complexes affecting yeast rRNA gene transcription. Using a mutant expressing non-degradable Rrn3p or a strain in which defined endogenous Rrn3p levels can be adjusted by the Tet-off system, we can demonstrate that Rrn3p levels influence the number of Pol I-Rrn3p complexes and consequently rRNA gene transcription. However, our analysis reveals that the dramatic reduction of rRNA synthesis in the immediate cellular response to impaired TOR signalling cannot be explained by the simple down-regulation of Rrn3p and Pol I-Rrn3p levels.

Site specific phosphorylation of yeast RNA polymerase I.

All nuclear RNA polymerases are phosphoprotein complexes. Yeast RNA polymerase I (Pol I) contains approximately 15 phosphate groups, distributed to 5 of the 14 subunits. Information about the function of the single phosphosites and their position in the primary, secondary and tertiary structure is lacking. We used a rapid and efficient way to purify yeast RNA Pol I to determine 13 phosphoserines and -threonines. Seven of these phosphoresidues could be located in the 3D-homology model for Pol I, five of them are more at the surface. The single phosphorylated residues were systematically mutated and the resulting strains and Pol I preparations were analyzed in cellular growth, Pol I composition, stability and genetic interaction with non-essential components of the transcription machinery. Surprisingly, all Pol I phosphorylations analyzed were found to be non-essential post-translational modifications. However, one mutation (subunit A190 S685D) led to higher growth rates in the presence of 6AU or under environmental stress conditions, and was synthetically lethal with a deletion of the Pol I subunit A12.2, suggesting a role in RNA cleavage/elongation or termination. Our results suggest that individual major or constitutively phosphorylated residues contribute to non-essential Pol I-functions.

Workplace ostracism, self-regulation, and job performance: Moderating role of intrinsic work motivation.

Drawing from a self-regulation perspective, we examine how intrinsic work motivation changes the relation between workplace ostracism and employee job performance via self-leadership. We test a moderated mediated model with data collected from 101 employees at two points in time. Results provide support for the hypothesis that ostracized employees who are more intrinsically motivated use self-leadership strategies to a greater degree to improve their job performance than their counterparts who are not intrinsically motivated. The findings contribute to research regarding boundary conditions of ostracism theory and have important practical implications.

Comparative characterization of all cellulosomal cellulases from Clostridium thermocellum reveals high diversity in endoglucanase product formation essential for complex activity.

BACKGROUND: Clostridium thermocellum is a paradigm for efficient cellulose degradation and a promising organism for the production of second generation biofuels. It owes its high degradation rate on cellulosic substrates to the presence of supra-molecular cellulase complexes, cellulosomes, which comprise over 70 different single enzymes assembled on protein-backbone molecules of the scaffold protein CipA. RESULTS: Although all 24 single-cellulosomal cellulases were described previously, we present the first comparative catalogue of all these enzymes together with a comprehensive analysis under identical experimental conditions, including enzyme activity, binding characteristics, substrate specificity, and product analysis. In the course of our study, we encountered four types of distinct enzymatic hydrolysis modes denoted by substrate specificity and hydrolysis product formation: (i) exo-mode cellobiohydrolases (CBH), (ii) endo-mode cellulases with no specific hydrolysis pattern, endoglucanases (EG), (iii) processive endoglucanases with cellotetraose as intermediate product (pEG4), and (iv) processive endoglucanases with cellobiose as the main product (pEG2). These modes are shown on amorphous cellulose and on model cello-oligosaccharides (with degree of polymerization DP 3 to 6). Artificial mini-cellulosomes carrying combinations of cellulases showed their highest activity when all four endoglucanase-groups were incorporated into a single complex. Such a modeled nonavalent complex (n = 9 enzymes bound to the recombinant scaffolding protein CipA) reached half of the activity of the native cellulosome. Comparative analysis of the protein architecture and structure revealed characteristics that play a role in product formation and enzyme processivity. CONCLUSIONS: The identification of a new endoglucanase type expands the list of known cellulase functions present in the cellulosome. Our study shows that the variety of processivities in the enzyme complex is a key enabler of its high cellulolytic efficiency. The observed synergistic effect may pave the way for a better understanding of the enzymatic interactions and the design of more active lignocellulose-degrading cellulase cocktails in the future.

Structure of the initiation-competent RNA polymerase I and its implication for transcription.

Eukaryotic RNA polymerase I (Pol I) is specialized in rRNA gene transcription synthesizing up to 60% of cellular RNA. High level rRNA production relies on efficient binding of initiation factors to the rRNA gene promoter and recruitment of Pol I complexes containing initiation factor Rrn3. Here, we determine the cryo-EM structure of the Pol I-Rrn3 complex at 7.5 Å resolution, and compare it with Rrn3-free monomeric and dimeric Pol I. We observe that Rrn3 contacts the Pol I A43/A14 stalk and subunits A190 and AC40, that association re-organizes the Rrn3 interaction interface, thereby preventing Pol I dimerization; and Rrn3-bound and monomeric Pol I differ from the dimeric enzyme in cleft opening, and localization of the A12.2 C-terminus in the active centre. Our findings thus support a dual role for Rrn3 in transcription initiation to stabilize a monomeric initiation competent Pol I and to drive pre-initiation complex formation.

Oleic acid is a precursor of linoleic acid and the male sex pheromone in Nasonia vitripennis.

Linoleic acid (C18:2(Δ9,12), LA) is crucial for many cell functions in organisms. It has long been a paradigm that animals are unable to synthesize LA from oleic acid (C18:1(Δ9), OA) because they were thought to miss Δ(12)-desaturases for inserting a double bound at the Δ(12)-position. Today it is clear that this is not true for all animals because some insects and other invertebrates have been demonstrated to synthesize LA. However, the ability to synthesize LA is known in only five insect orders and no examples have been reported so far in the Hymenoptera. LA plays a particular role in the parasitic wasp Nasonia vitripennis, because it is the precursor of the male sex pheromone consisting of (4R,5R)- and (4R,5S)-5-hydroxy-4-decanolides. Here we demonstrate by stable isotope labeling that N. vitripennis is able to incorporate externally applied fully (13)C-labeled OA into the male sex pheromone suggesting that they convert initially OA into LA. To verify this assumption, we produced fly hosts (Lucilia caesar) which were experimentally enriched in (13)C-labeled OA and reared male parasitoids on these hosts. Chemical analysis of transesterified lipid raw extracts from hosts and parasitoids revealed that N. vitripennis but not L. caesar contained (13)C-labeled LA methyl ester. Furthermore, male wasps from the manipulated hosts produced significant amounts of (13)C-labeled sex pheromone. These results suggest that N. vitripennis possesses a Δ(12)-desaturase. The additional fitness relevant function as pheromone precursor might have favored the evolution of LA biosynthesis in N. vitripennis to make the wasps independent of the formerly essential nutrient.

Reduction in ribosomal protein synthesis is sufficient to explain major effects on ribosome production after short-term TOR inactivation in Saccharomyces cerevisiae.

Ribosome synthesis depends on nutrient availability, sensed by the target of rapamycin (TOR) signaling pathway in eukaryotes. TOR inactivation affects ribosome biogenesis at the level of rRNA gene transcription, expression of ribosomal proteins (r-proteins) and biogenesis factors, preribosome processing, and transport. Here, we demonstrate that upon TOR inactivation, levels of newly synthesized ribosomal subunits drop drastically before the integrity of the RNA polymerase I apparatus is severely impaired but in good correlation with a sharp decrease in r-protein production. Inhibition of translation by cycloheximide mimics the rRNA maturation defect observed immediately after TOR inactivation. Both cycloheximide addition and the depletion of individual r-proteins also reproduce TOR-dependent nucleolar entrapment of specific ribosomal precursor complexes. We suggest that shortage of newly synthesized r-proteins after short-term TOR inactivation is sufficient to explain most of the observed effects on ribosome production.