RNA-binding proteins play a key role in shaping gene expression profiles during stress, however, little is known about the dynamic nature of these interactions and how this influences the kinetics of ...gene expression. To address this, we developed kinetic cross-linking and analysis of cDNAs (χCRAC), an ultraviolet cross-linking method that enabled us to quantitatively measure the dynamics of protein-RNA interactions in vivo on a minute time-scale. Here, using χCRAC we measure the global RNA-binding dynamics of the yeast transcription termination factor Nab3 in response to glucose starvation. These measurements reveal rapid changes in protein-RNA interactions within 1 min following stress imposition. Changes in Nab3 binding are largely independent of alterations in transcription rate during the early stages of stress response, indicating orthogonal transcriptional control mechanisms. We also uncover a function for Nab3 in dampening expression of stress-responsive genes. χCRAC has the potential to greatly enhance our understanding of in vivo dynamics of protein-RNA interactions.Protein RNA interactions are dynamic and regulated in response to environmental changes. Here the authors describe 'kinetic CRAC', an approach that allows time resolved analyses of protein RNA interactions with minute time point resolution and apply it to gain insight into the function of the RNA-binding protein Nab3.
TOR kinase complex I (TORC1) is a key regulator of cell growth and metabolism in all eukaryotes. Previous studies in yeast have shown that three GTPases-Gtr1, Gtr2, and Rho1-bind to TORC1 in nitrogen ...and amino acid starvation conditions to block phosphorylation of the S6 kinase Sch9 and activate protein phosphatase 2A (PP2A). This leads to downregulation of 450 Sch9-dependent protein and ribosome synthesis genes and upregulation of 100 PP2A-dependent nitrogen assimilation and amino acid synthesis genes. Here, using bandshift assays and microarray measurements, we show that the TORC1 pathway also populates three other stress/starvation states. First, in glucose starvation conditions, the AMP-activated protein kinase (AMPK/Snf1) and at least one other factor push the TORC1 pathway into an off state, in which Sch9-branch signaling and PP2A-branch signaling are both inhibited. Remarkably, the TORC1 pathway remains in the glucose starvation (PP2A inhibited) state even when cells are simultaneously starved for nitrogen and glucose. Second, in osmotic stress, the MAPK Hog1/p38 drives the TORC1 pathway into a different state, in which Sch9 signaling and PP2A-branch signaling are inhibited, but PP2A-branch signaling can still be activated by nitrogen starvation. Third, in oxidative stress and heat stress, TORC1-Sch9 signaling is blocked while weak PP2A-branch signaling occurs. Together, our data show that the TORC1 pathway acts as an information-processing hub, activating different genes in different conditions to ensure that available energy is allocated to drive growth, amino acid synthesis, or a stress response, depending on the needs of the cell.
Pathway optimization plays an important role in fine-tuning metabolic pathways. In most conditions, more than three genes are involved in the biosynthesis pathway of a specific target product. To ...improve the titer of products, rational regulation of a group of genes by a series of promoters with different strengths is essential. On the basis of a series of RNA-Seq data, a set of 66 native promoters was chosen to fine-tune gene expression in Saccharomyces cerevisiae. Promoter strength was characterized by measuring the fluorescence strength of the enhanced green fluorescent protein through fluorescence-activated cell sorting. The expressions of PTDH1, PPGK1, PINO1, PSED1, and PCCW12 were stronger than that of PTDH3, whereas those of another 15 promoters were stronger than that of PTEF1. Then, 30 promoters were chosen to optimize the biosynthesis pathway of (2S)-naringenin from p-coumaric acid. With a high-throughput screening method, the highest titer of (2S)-naringenin in a 5 L bioreactor reached 1.21 g/L from p-coumaric acid, which is the highest titer according to the currently available reports.
In this study, bioethanol production from steam-exploded wheat straw using different process configurations was evaluated using two Saccharomyces cerevisiae strains, F12 and Red Star. The strain F12 ...has been engineerically modified to allow xylose consumption as cereal straw contain considerable amounts of pentoses. Red Star is a robust hexose-fermenting strain used for industrial fuel ethanol fermentations and it was used for comparative purposes. The highest ethanol concentration, 23.7 g/L, was reached using the whole slurry (10%, w/v) and the recombinant strain (F12) in an SSF process, it showed an ethanol yield on consumed sugars of 0.43 g/g and a volumetric ethanol productivity of 0.7 g/L h for the first 3 h. Ethanol concentrations obtained in SSF processes were in all cases higher than those from SHF at the same conditions. Furthermore, using the whole slurry, final ethanol concentration was improved in all tests due to the increase of potential fermentable sugars in the fermentation broth. Inhibitory compounds present in the pretreated wheat straw caused a significantly negative effect on the fermentation rate. However, it was found that the inhibitors furfural and HMF were completely metabolized by the yeast during SSF by metabolic redox reactions. An often encountered problem during xylose fermentation is considerable xylitol production that occurs due to metabolic redox imbalance. However, in our work this redox imbalance was counteracted by the detoxification reactions and no xylitol was produced. Biotechnol. Bioeng. 2008;100: 1122-1131.
The endosomal system functions as a network of protein and lipid sorting stations that receives molecules from endocytic and secretory pathways and directs them to the lysosome for degradation, or ...exports them from the endosome via retrograde trafficking or plasma membrane recycling pathways. Retrograde trafficking pathways describe endosome‐to‐Golgi transport while plasma membrane recycling pathways describe trafficking routes that return endocytosed molecules to the plasma membrane. These pathways are crucial for lysosome biogenesis, nutrient acquisition and homeostasis and for the physiological functions of many types of specialized cells. Retrograde and recycling sorting machineries of eukaryotic cells were identified chiefly through genetic screens using the budding yeast Saccharomyces cerevisiae system and discovered to be highly conserved in structures and functions. In this review, we discuss advances regarding retrograde trafficking and recycling pathways, including new discoveries that challenge existing ideas about the organization of the endosomal system, as well as how these pathways intersect with cellular homeostasis pathways.
Retrograde trafficking and plasma membrane recycling pathways originating from the endosome serve many physiological functions, including facilitating the reuse of sorting receptors in organelle biogenesis pathways and controlling plasma membrane composition. Genetic studies using Saccharomyces cerevisiae first identified machineries involved in retrograde and recycling sorting pathways, many of which are highly conserved. Recent advances in yeast give insight into how these pathways intersect with cellular homeostasis pathways and challenge existing ideas about the organization of the endosomal system.
Eukaryotic DNA replication is initiated at multiple origins of replication, where many replication proteins assemble under the control of the cell cycle 1. A key process of replication initiation is ...to convert inactive Mcm2–7 to active Cdc45-Mcm-GINS (CMG) replicative helicase 2. However, it is not known whether the CMG assembly would automatically activate its helicase activity and thus assemble the replisome. Mcm10 is an evolutionally conserved essential protein required for the initiation of replication 3, 4. Although the roles of many proteins involved in the initiation are understood, the role of Mcm10 remains controversial 5–9. To characterize Mcm10 in more detail, we constructed budding yeast cells bearing a degron-fused Mcm10 protein that can be efficiently degraded in response to auxin. In the absence of Mcm10, a stable CMG complex was assembled at origins. However, subsequent translocation of CMG, replication protein A loading to origins, and the intra-S checkpoint activation were severely diminished, suggesting that origin unwinding is defective. We also found that Mcm10 associates with origins during initiation in an S-cyclin-dependent kinase- and Cdc45-dependent manner. Thus, Mcm10 plays an essential role in functioning of the CMG replicative helicase independent of assembly of a stable CMG complex at origins.
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► A stable CMG complex stays at origins without Mcm10 ► Origin unwinding is defective in the absence of Mcm10 ► Our study predicts that Mcm10 is involved in activation of CMG ► Mcm10 associates with origins in an S-CDK- and Cdc45-dependent manner
Eukaryotic chromosomes are replicated from multiple origins that initiate throughout the S‐phase of the cell cycle. Why all origins do not fire simultaneously at the beginning of S‐phase is not ...known, but two kinase activities, cyclin‐dependent kinase (CDK) and Dbf4‐dependent kinase (DDK), are continually required throughout the S‐phase for all replication initiation events. Here, we show that the two CDK substrates Sld3 and Sld2 and their binding partner Dpb11, together with the DDK subunit Dbf4 are in low abundance in the budding yeast, Saccharomyces cerevisiae. Over‐expression of these factors is sufficient to allow late firing origins of replication to initiate early and together with deletion of the histone deacetylase RPD3, promotes the firing of heterochromatic, dormant origins. We demonstrate that the normal programme of origin firing prevents inappropriate checkpoint activation and controls S‐phase length in budding yeast. These results explain how the competition for limiting DDK kinase and CDK targets at origins regulates replication initiation kinetics during S‐phase and establishes a unique system with which to investigate the biological roles of the temporal programme of origin firing.
Early‐firing replication origins appear to out‐compete late‐firing origins for certain key initiation factors. The resulting stepwise progression of genome duplication helps to maintain sufficient nucleotide pools throughout the S‐phase.
The 25S rRNA of yeast contains several base modifications in the functionally important regions. The enzymes responsible for most of these base modifications remained unknown. Recently, we identified ...Rrp8 as a methyltransferase involved in m(1)A645 modification of 25S rRNA. Here, we discovered a previously uncharacterized gene YBR141C to be responsible for second m(1)A2142 modification of helix 65 of 25S rRNA. The gene was identified by reversed phase-HPLC screening of all deletion mutants of putative RNA methyltransferase and was confirmed by gene complementation and phenotypic characterization. Because of the function of its encoded protein, YBR141C was named BMT2 (base methyltransferase of 25S RNA). Helix 65 belongs to domain IV, which accounts for most of the intersubunit surface of the large subunit. The 3D structure prediction of Bmt2 supported it to be an Ado Met methyltransferase belonging to Rossmann fold superfamily. In addition, we demonstrated that the substitution of G180R in the S-adenosyl-L-methionine-binding motif drastically reduces the catalytic function of the protein in vivo. Furthermore, we analysed the significance of m(1)A2142 modification in ribosome synthesis and translation. Intriguingly, the loss of m(1)A2142 modification confers anisomycin and peroxide sensitivity to the cells. Our results underline the importance of RNA modifications in cellular physiology.
Ribosome assembly in eukaryotes requires approximately 200 essential assembly factors (AFs) and occurs through ordered events that initiate in the nucleolus and culminate in the cytoplasm. Here, we ...present the electron cryo-microscopy (cryo-EM) structure of a late cytoplasmic 40S ribosome assembly intermediate from Saccharomyces cerevisiae at 18 angstrom resolution. We obtained cryo-EM reconstructions of preribosomal complexes lacking individual components to define the positions of all seven AFs bound to this intermediate. These late-binding AFs are positioned to prevent each step in the translation initiation pathway. Together, they obstruct the binding sites for initiation factors, prevent the opening of the messenger RNA channel, block 60S subunit joining, and disrupt the decoding site. These redundant mechanisms probably ensure that pre-40S particles do not enter the translation pathway, which would result in their rapid degradation.
Secondary structure-forming DNA sequences such as CAG repeats interfere with replication and repair, provoking fork stalling, chromosome fragility, and recombination. In budding yeast, we found that ...expanded CAG repeats are more likely than unexpanded repeats to localize to the nuclear periphery. This positioning is transient, occurs in late S phase, requires replication, and is associated with decreased subnuclear mobility of the locus. In contrast to persistent double-stranded breaks, expanded CAG repeats at the nuclear envelope associate with pores but not with the inner nuclear membrane protein Mps3. Relocation requires Nup84 and the Slx5/8 SUMO-dependent ubiquitin ligase but not Rad51, Mec1, or Tel1. Importantly, the presence of the Nup84 pore subcomplex and Slx5/8 suppresses CAG repeat fragility and instability. Repeat instability in nup84, slx5, or slx8 mutant cells arises through aberrant homologous recombination and is distinct from instability arising from the loss of ligase 4-dependent end-joining. Genetic and physical analysis of Rad52 sumoylation and binding at the CAG tract suggests that Slx5/8 targets sumoylated Rad52 for degradation at the pore to facilitate recovery from acute replication stress by promoting replication fork restart. We thereby confirmed that the relocation of damage to nuclear pores plays an important role in a naturally occurring repair process.
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