Ribosomal RNA (rRNA) transcription is regulated primarily at the level of initiation from rRNA promoters. The unusual kinetic properties of these promoters result in their specific regulation by two small molecule signals, ppGpp and the initiating NTP, that bind to RNA polymerase (RNAP) at all promoters. We show here that DksA, a protein previously unsuspected as a transcription factor, is absolutely required for rRNA regulation. In deltadksA mutants, rRNA promoters are unresponsive to changes in amino acid availability, growth rate, or growth phase. In vitro, DksA binds to RNAP, reduces open complex lifetime, inhibits rRNA promoter activity, and amplifies effects of ppGpp and the initiating NTP on rRNA transcription, explaining the dksA requirement in vivo. These results expand our molecular understanding of rRNA transcription regulation, may explain previously described pleiotropic effects of dksA, and illustrate how transcription factors that do not bind DNA can nevertheless potentiate RNAP for regulation.

To establish and maintain organ structure and function, tissues need to balance stem cell proliferation and differentiation rates and coordinate cell fate with position. By quantifying and modelling tissue stress and deformation in the mammalian epidermis, we find that this balance is coordinated through local mechanical forces generated by cell division and delamination. Proliferation within the basal stem/progenitor layer, which displays features of a jammed, solid-like state, leads to crowding, thereby locally distorting cell shape and stress distribution. The resulting decrease in cortical tension and increased cell-cell adhesion trigger differentiation and subsequent delamination, reinstating basal cell layer density. After delamination, cells establish a high-tension state as they increase myosin II activity and convert to E-cadherin-dominated adhesion, thereby reinforcing the boundary between basal and suprabasal layers. Our results uncover how biomechanical signalling integrates single-cell behaviours to couple proliferation, cell fate and positioning to generate a multilayered tissue.

Previous investigations into the mechanisms that control RNA Polymerase (Pol) I transcription have primarily focused on the process of transcription initiation, thus little is known regarding postinitiation steps in the transcription cycle. Spt4p and Spt5p are conserved throughout eukaryotes, and they affect elongation by Pol II. We have found that these two proteins copurify with Pol I and associate with the rDNA in vivo. Disruption of the gene for Spt4p resulted in a modest decrease in growth and rRNA synthesis rates at the permissive temperature, 30°C. Furthermore, biochemical and EM analyses showed clear defects in rRNA processing. These data suggest that Spt4p, Spt5p, and, potentially, other regulators of Pol I transcription elongation play important roles in coupling rRNA transcription to its processing and ribosome assembly.yeast Saccharomyces cerevisiae T he synthesis of ribosomal RNA (rRNA) by RNA Polymerase (Pol) I is an important step in the synthesis of ribosomes, and its regulation is closely linked to the nutrient conditions and growth potential for the cell. Previous studies have identified essential components of the Pol I transcription apparatus as well as important cis-elements in Pol I promoters and revealed some potential mechanisms for regulation of transcription initiation (for reviews, see refs. 1-5). Unlike Pol II, however, little is known regarding mechanisms that regulate postinitiation steps of transcription by Pol I.One well characterized Pol II transcription elongation factor in yeast is a complex of two proteins, Spt4p and Spt5p (the complex will be referred to as Spt4͞5 here). The SPT4 and SPT5 genes were among many genes isolated for their ability to suppress transcription defects caused by insertions of the retrotransposon Ty1 (or the Ty1 long terminal repeats, ␦) in the 5Ј noncoding regions of yeast genes (6). Swanson and Winston (7) later showed that Spt4͞5 associates with Spt6p, which affects Pol II elongation through chromatin. However, Spt4p and Spt5p also form a separate complex, devoid of Spt6p, that associates with Pol II physically and genetically, and this interaction is important for transcription elongation (8). More recent work has shown that deletion of the nonessential gene SPT4 results in reduced efficiency of Pol II elongation through GC-rich DNA sequences (9) and a general decrease in Pol II processivity (10). Taken together, all of these data clearly support a role for Spt4͞5 in transcription elongation by Pol II in yeast.Spt4͞5 also plays a role in Pol II transcription elongation in mammalian cells. The mammalian homologues of Spt4p and Spt5p form a complex called the 5,6-dichloro-1-␤-Dribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF), which was originally identified as a factor that induces a DRB-dependent arrest of elongating Pol II complexes in reconstituted in vitro transcription assays (11). It was demonstrated that under nucleotide-limiting conditions, DSIF could also increase the rate of elongation of Pol II in vitro. Thus, work in mam...

Previous studies showed that adenosine triphosphate (ATP) concentrations in Escherichia coli changed during certain growth transitions and directly controlled the rate of rRNA transcription initiation at those times. The relationship between ATP concentration and rRNA transcription during steady-state growth is less clear, however. This is because two commonly employed methods for measuring ATP concentrations in bacteria, both of which rely on physical extraction followed by chromatographic separation of small molecules, resulted in dramatically different conclusions about whether ATP concentration changed with steady-state growth rate. Extraction with formic acid indicated that ATP concentration did not change with growth rate, whereas formaldehyde treatment followed by extraction with alkali indicated that ATP concentration increased proportionally to the growth rate. To resolve this discrepancy, we developed a bioassay for ATP based on the expression of a variant of the firefly luciferase enzyme in vivo and measurement of luminescence in cells growing in different conditions. We found that the available ATP concentration did not vary with growth rate, either in wildtype cells or in cells lacking guanosine 5-diphosphate, 3-diphosphate, providing insight into the regulation of rRNA transcription. More broadly, the luciferase bioassay described here provides a general method for evaluating the ATP concentration available for biochemical processes in E. coli and potentially in other organisms.ATP is the energy currency of the cell, providing energy for enzymatic reactions. ATP also serves as a substrate for RNA synthesis, and it regulates a variety of biological processes. Because of its potential to affect many aspects of cellular regulation, whether cellular ATP concentrations change when growth conditions change is a key question in molecular biology.The concentration of the initiating nucleoside triphosphate (iNTP) 1 plays a direct role in the regulation of rRNA transcription initiation in Escherichia coli in response to changes in growth conditions (1-4). For example, when cells outgrow from stationary phase, the levels of ATP (the iNTP for 6 rrn P1 promoters) and GTP (the iNTP for the seventh rrn P1 promoter) increase dramatically, resulting in a direct and rapid increase in rRNA synthesis (4). Conversely, when cells enter stationary phase, a decrease in ATP and GTP levels (in conjunction with an increase in the concentration of guanosine 5Ј-diphosphate, 3Ј-diphosphate, ppGpp) directly inhibits rRNA synthesis. Following nutrient shifts during exponential growth, changes in ppGpp concentration are sufficient to account for regulation of rRNA promoters (4).It has long been known that when E. coli cells are grown on different nutrient sources leading to different steady-state growth rates rRNA synthesis is proportional to the square of the culture's growth rate (5). The molecular basis for this phenomenon, called "growth rate-dependent control", still remains unresolved, however. Based on an observed correlati...

Videocapsule endoscopy shows good sensitivity and excellent specificity for the detection of villous atrophy in patients with suspected celiac disease.

The synthesis of ribosomes in eukaryotic cells is a complex process involving many nonribosomal protein factors and snoRNAs. In general, the processes of rRNA transcription and ribosome assembly are treated as temporally or spatially distinct. Here, we describe the identification of a point mutation in the second largest subunit of RNA polymerase I near the active center of the enzyme that results in an elongation-defective enzyme in the yeast Saccharomyces cerevisiae. In vivo, this mutant shows significant defects in rRNA processing and ribosome assembly. Taken together, these data suggest that transcription of rRNA by RNA polymerase I is linked to rRNA processing and maturation. Thus, RNA polymerase I, elongation factors, and rRNA sequence elements appear to function together to optimize transcription elongation, coordinating cotranscriptional interactions of many factors/snoRNAs with pre-rRNA for correct rRNA processing and ribosome assembly.

A cornerstone of preclinical cancer research has been the use of clonal cell lines. However, this resource has underperformed in its ability to effectively identify novel therapeutics and evaluate the heterogeneity in a patient's tumor. The patient-derived xenograft (PDX) model retains the heterogeneity of patient tumors, allowing a means to not only examine efficacy of a therapy, but also basic tenets of cancer biology in response to treatment. Herein we describe the development and characterization of an ovarian-PDX model in order to study the development of chemoresistance. We demonstrate that PDX tumors are not simply composed of tumor-initiating cells, but recapitulate the original tumor's heterogeneity, oncogene expression profiles, and clinical response to chemotherapy. Combined carboplatin/paclitaxel treatment of PDX tumors enriches the cancer stem cell populations, but persistent tumors are not entirely composed of these populations. RNA-Seq analysis of six pair of treated PDX tumors compared to untreated tumors demonstrates a consistently contrasting genetic profile after therapy, suggesting similar, but few, pathways are mediating chemoresistance. Pathways and genes identified by this methodology represent novel approaches to targeting the chemoresistant population in ovarian cancer

Chromosome condensation and the global repression of gene transcription1 are features of mitosis in most eukaryotes. The logic behind this phenomenon is that chromosome condensation prevents the activity of RNA polymerases. In budding yeast, however, transcription was proposed to be continuous during mitosis2. Here we show that Cdc14, a protein phosphatase required for nucleolar segregation3 and mitotic exit4, inhibits transcription of yeast ribosomal genes (rDNA) during anaphase. The phosphatase activity of Cdc14 is required for RNA polymerase I (Pol I) inhibition in vitro and in vivo. Moreover Cdc14-dependent inhibition involves nucleolar exclusion of Pol I subunits. We demonstrate that transcription inhibition is necessary for complete chromosome disjunction, because ribosomal RNA (rRNA) transcripts block condensin binding to rDNA, and show that bypassing the role of Cdc14 in nucleolar segregation requires in vivo degradation of nascent transcripts. Our results show that transcription interferes with chromosome condensation, not the reverse. We conclude that budding yeast, like most eukaryotes, inhibit Pol I transcription before segregation as a prerequisite for chromosome condensation and faithful genome separation.