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This paper studied the rock dynamic fracture propagation under impact loads elaborately with a determination method proposed to calculate crack propagation dynamic stress intensity factor (DSIF). By utilizing the split-Hopkinson pressure bar, the impact experiments with an improved single cleavage semi-circle (ISCSC) specimen were conducted to illuminate the dynamic crack propagation behaviour. Meanwhile, the fracture characteristics and crack propagation velocity were obtained by the crack propagation gauges. Coordinating experiments with a numerical approach, the crack propagation dynamic stress intensity factors were calculated by an experimental–numerical method with fractal theory. Then, a finite difference model was developed based on the tensile fracture softening damage criterion. With the analysis of numerical and experimental results, the crack propagation behaviour and mechanism of crack arrest were discussed sophisticatedly. The results demonstrate that the novel ISCSC specimen shows a definite advantage in determining crack propagation and arrest DSIF. Additionally, the crack arrest DSIF is larger than the average propagation DSIF with a sharp increase. Meanwhile, the numerical simulation results which agree well with the actual crack propagation illustrate that the crack arrest should be dominated by the compressive stress perpendicular to the crack path, and there were several arrest pauses existing in the transitory crack arrest process.

Transcriptional pausing is a widely used regulatory mechanism to control precise gene expression from bacteria to humans. In metazoans, transcription initiation requires the recruitment of general transcription factors and RNA polymerase II (Pol II) to gene promoters. After it initiates transcription and synthesizes a short RNA, Pol II pauses at the promoter-proximal region, standing by for further cues to enter the productive elongation stage. Promoter-proximal Pol II pausing is one of the rate-limiting steps during transcription, functioning as an early transcriptional elongation check point, to ensure the correct modification of transcription machinery and the addition of the 7-methylguanosine cap to nascent transcripts. Pol II pausing at promoter-proximal region is stabilized by the negative elongation factor (NELF) and the 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF). In response to developmental signals or environmental stress, the release of paused Pol II can be triggered by the positive transcription elongation factor b (P-TEFb), which is composed of the kinase CDK9 and its regulatory subunit CCNT1/2. The kinase activity of P-TEFb towards Pol II carboxy-terminal domain (CTD), NELF and DSIF is critical for P-TEFb to execute its function in pause release, and is thus tightly controlled. To date, it has been identified that P-TEFb mainly exists in three different complexes: the super elongation complex (SEC), BRD4-P-TEFb, and the inactive 7SK snRNP-P-TEFb complex. SEC, one of the most active P-TEFb containing complexes, is required for rapid gene expression in response to stress or developmental signals. The active and inactive forms of P-TEFb complexes co-exist in vivo , reaching equilibrium to meet the transcriptional needs of cells. The transition of Pol II from pause to productive elongation stage is achieved by dynamic exchange between the pausing factors and the positive elongation factors at the proximal promoter region. Liquid-liquid phase separation (LLPS) in living cells is a physicochemical process that confines biochemical reactions within membrane-less compartments. Low complexity intrinsically disordered regions (IDRs) within proteins can initiate and promote phase separation and formation of liquid droplets, which in turn facilitate the multivalent weak interactions among these IDR-containing proteins. Recent studies using super-resolution live cell imaging technology have revealed that RNA Pol II can form dynamic foci in cells through LLPS and transitions between different phases, which are likely mediated by phosphorylated CTD. Moreover, factors involved in various transcription steps can rapidly and dynamically concentrate and exchange at the hot regulatory genomic loci. For example, SEC components are able to compartmentalize and concentrate P-TEFb from its inactive partners. Upon serum stimulation, SEC assembles promptly into phase-separated droplets at the immediate early genes (IEG), triggering rapid transcription induction. Mutations in transcription regulators can lead to developmental defects and many human diseases, including cancers. For instance, abnormal stabilization of SEC components via MLL fusion plays a contributing role in leukemogenesis. Fusion of the SEC core component ENL with MLL can greatly enhance phase separation of SEC, through which the leukemic genes might be mis-activated. In addition, there is growing evidence that disease related mutations in transcriptional regulators can cause malfunction through aberrant condensation, providing potential targets for designing therapeutic strategies through phase separation interventions.

Activation-induced deoxycytidine deaminase (AID) initiates somatic hypermutation (SHM) in immunoglobulin variable (IgV) genes to produce high-affinity antibodies. SHM requires IgV transcription by RNA polymerase II (Pol II). A eukaryotic transcription system including AID has not been reported previously. Here, we reconstitute AID-catalyzed deamination during Pol II transcription elongation in conjunction with DSIF transcription factor. C→T mutations occur at similar frequencies on non-transcribed strand (NTS) and transcribed strand (TS) DNA. In contrast, bacteriophage T7 Pol generates NTS mutations predominantly. AID-Pol II mutations are strongly favored in WRC and WGCW overlapping hot motifs (W = A or T, R = A or G) on both DNA strands. Single mutations occur on 70% of transcribed DNA clones. Mutations are correlated over a 15 nt distance in multiply mutated clones, suggesting that deaminations are catalyzed processively within a stalled or backtracked transcription bubble. Site-by-site comparisons for biochemical and human memory B-cell mutational spectra in an IGHV3-23*01 target show strongly favored deaminations occurring in the antigen-binding complementarity determining regions (CDR) compared to the framework regions (FW). By exhibiting consistency with B-cell SHM, our in vitro data suggest that biochemically defined reconstituted Pol II transcription systems can be used to investigate how, when and where AID is targeted.

Control of gene expression is one of the most complex yet continuous physiological processes impacting cellular homeostasis. RNA polymerase II (Pol II) transcription is tightly regulated at promoter-proximal regions by intricate dynamic processes including Pol II pausing, release into elongation and premature termination. Pol II pausing is a phenomenon where Pol II complex pauses within 30–60 nucleotides after initiating the transcription. Negative elongation factor (NELF) and DRB sensitivity inducing factor (DSIF) contribute in the establishment of Pol II pausing, and positive transcription elongation factor b releases (P-TEFb) paused complex after phosphorylating DSIF that leads to dissociation of NELF. Pol II pausing is observed in most expressed genes across the metazoan. The precise role of Pol II pausing is not well understood; however, it’s required for integration of signals for gene regulation. In the present study, we investigated the role of phosphatase and tensin homolog (PTEN) in genome-wide transcriptional regulation using PTEN overexpression and PTEN knock-down models. Here we identify that PTEN alters the expression of hundreds of genes, and its restoration establishes genome-wide Pol II promoter-proximal pausing in PTEN null cells. Furthermore, PTEN re-distributes Pol II occupancy across the genome and possibly impacts Pol II pause duration, release and elongation rate in order to enable precise gene regulation at the genome-wide scale. Our observations demonstrate an imperative role of PTEN in global transcriptional regulation that will provide a new direction to understand PTEN-associated pathologies and its management.

Gene expression by RNA polymerase II (RNAPII) is tightly controlled by cyclin-dependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The pausing checkpoint following transcription initiation is primarily controlled by CDK9. We discovered that CDK9-mediated, RNAPII-driven transcription is functionally opposed by a protein phosphatase 2A (PP2A) complex that is recruited to transcription sites by the Integrator complex subunit INTS6. PP2A dynamically antagonizes phosphorylation of key CDK9 substrates including DSIF and RNAPII-CTD. Loss of INTS6 results in resistance to tumor cell death mediated by CDK9 inhibition, decreased turnover of CDK9 phospho-substrates, and amplification of acute oncogenic transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill both leukemic and solid tumor cells, providing therapeutic benefit in vivo. These data demonstrate that fine control of gene expression relies on the balance between kinase and phosphatase activity throughout the transcription cycle, a process dysregulated in cancer that can be exploited therapeutically.

RNA polymerases (Pols) I, II, and III collectively synthesize most of the RNA in a eukaryotic cell. Transcription by Pols I, II, and III is regulated by hundreds of trans-acting factors. One such protein, Spt4, has been previously identified as a transcription factor that influences both Pols I and II. Spt4 forms a complex with Spt5, described as the Spt4/5 complex (or DSIF in mammalian cells). This complex has been shown previously to directly interact with Pol I and potentially affect transcription elongation. The previous literature identified defects in transcription by Pol I when SPT4 was deleted, but the necessary tools to characterize the mechanism of this effect were not available at the time. Here, we use a technique called Native Elongating Transcript Sequencing (NET-seq) to probe for the global occupancy of Pol I in wild-type (WT) and spt4△ Saccharomyces cerevisiae (yeast) cells at single nucleotide resolution in vivo. Analysis of NET-seq data reveals that Spt4 promotes Pol I processivity and enhances transcription elongation through regions of the ribosomal DNA that are particularly G-rich. These data suggest that Spt4/5 may directly affect transcription elongation by Pol I in vivo.

Regulation of transcription is a tightly choreographed process. The establishment of RNA polymerase II promoter proximal pausing soon after transcription initiation and the release of Pol II into productive elongation are key regulatory processes that occur in early elongation. We describe the techniques and tools that have become available for the study of promoter proximal pausing and their utility for future experiments. We then provide an overview of the factors and interactions that govern a multipartite pausing process and address emerging questions surrounding the mechanism of RNA polymerase II's subsequent advancement into the gene body. Finally, we address remaining controversies and future areas of study.

Starenje dovodi do progresivnog narušavanja homeostaze organizma, te posljedično povećanog rizika obolijevanja od različitih bolesti povezanih sa starenjem. Pravilna ekspresija gena ključna je za stanični identitet, pa su stoga promjene regulacije ekspresije vjerojatno jedan od prvih događaja narušenih starenjem. Posljednji korak kompleksnog procesa regulacije transkripcije gena - prijelaz RNA polimeraze II (Pol II) iz koraka inicijacije u elongaciju - karakterizira pauziranje Pol II 20-60 pb nizvodno od mjesta početka transkripcije. Tijekom starenja dolazi do globalnog smanjenja ovakvog pauziranja proksimalno od promotora. U ovom diplomskom radu pratila sam promjene u ekspresiji gena povezane sa starenjem, te u vezanju transkripcijskih faktora koji reguliraju pauziranje Pol II u jetri miša: Spt4, NELF-A, PAF1, Spt16, Spt6. Analiza ekspresije transkripcijskih faktora pokazala je smanjenje razine Spt16, podjedinice kompleksa FACT koji olakšava prolaz Pol II kroz nukleosome, povezano sa starenjem. Kako bih pratila promjene u vezanju transkripcijskih faktora u promotorskoj regiji gena, uspostavila sam protokol za kromatinsku imunoprecipitaciju popraćenu PCR metodom u stvarnom vremenu (ChIP-qPCR), u mišjoj jetri. Eksperimenti ChIP-qPCR na odabranom lokusu Hsf1 pokazali su smanjenje vezanja pauzirajućeg faktora Spt4 koji stabilizira pauzirani Pol II kompleks. Ovaj rezultat upućuje na efikasnije otpuštanje pauzirane Pol II u produktivnu elongaciju i u skladu je sa zabilježenim smanjenjem pauziranja Pol II proksimalno od promotora koje se dešava tijekom starenja. Nadalje, u ovom radu sam priredila i biblioteku sekvenci za analizu metodom ChIP-seq koja bi mogla dati pouzdanije podatke o smanjenju vezanja Spt4 ovisno o starenju. Ageing leads to the progressive impairment of homeostasis and is secondary to multiple aberrant processes. Proper gene regulation is a key to cellular identity, therefore an aberrant change in gene expression is likely one of the initial processes associated with ageing. Regulation of transcription is a complex process. The final regulatory step – the transition from initiation to elongation of RNA Polymerase II (Pol II) – is characterized by Pol II pausing 20-60 bp downstream of the transcription start site. This promoter-proximal pausing of Pol II is demonstrated to globally decrease during ageing. Here, I monitor age-related changes in expression and recruitment of the transcription factors that regulate Pol II pausing in murine liver: Spt4, NELF-A, PAF1, Spt16, Spt6. Western blot analysis of transcription factor expression patterns revealed an age-related decrease in Spt16 abundance, a subunit of the FACT complex that facilitates Pol II passage through nucleosomes. For monitoring the recruitment of transcription factors to gene promoters, chromatin immunoprecipitation followed by qPCR (ChIP-qPCR) was performed after establishing a ChIP protocol for murine liver tissue. ChIPqPCR experiments at an exemplary locus Hsf1 demonstrated an age-related decrease in the recruitment of Spt4, a pausing factor that stabilizes the paused Pol II complex. This observation suggests a more efficient Pol II pause release with age and is in line with the observed age-related decrease in promoterproximal Pol II pausing. Additionally, libraries for ChIP-seq data analysis, which is expected to confirm the age-related decrease in Spt4 recruitment on a genome-wide level, have been established in this work. (

For many years it was believed that promoter-proximal RNA-polymerase II (Pol II) pausing manages the transcription of genes in Drosophila development by controlling spatiotemporal properties of their activation and repression. But the exact proteins that cooperate to stall Pol II in promoter-proximal regions of developmental genes are still largely unknown. The current work describes the molecular mechanism employed by the Negative ELongation Factor (NELF) to control the Pol II pause at genes whose transcription is induced by 20-hydroxyecdysone (20E). According to our data, the NELF complex is recruited to the promoters and enhancers of 20E-dependent genes. Its presence at the regulatory sites of 20E-dependent genes correlates with observed interaction between the NELF-A subunit and the ecdysone receptor (EcR). The complete NELF complex is formed at the 20E-dependent promoters and participates in both their induced transcriptional response and maintenance of the uninduced state to keep them ready for the forthcoming transcription. NELF depletion causes a significant decrease in transcription induced by 20E, which is associated with the disruption of Pol II elongation complexes. A considerable reduction in the promoter-bound level of the Spt5 subunit of transcription elongation factor DSIF was observed at the 20E-dependent genes upon NELF depletion. We presume that an important function of NELF is to participate in stabilizing the Pol II-DSIF complex, resulting in a significant impact on transcription of its target genes. In order to directly link NELF to regulation of 20E-dependent genes in development, we show the presence of NELF at the promoters of 20E-dependent genes during their active transcription in both embryogenesis and metamorphosis. We also demonstrate that 20E-dependent promoters, while temporarily inactive at the larval stage, preserve a Pol II paused state and bind NELF complex.

Protein phosphatase 1 complex (PP1C), consisting of one of the phosphatases, PP1α, β and γ, and three regulatory subunits, PNUTS, TOX4 and WDR82, plays critical roles in gene regulation and is mutated in over 20% of uterine corpus endometrial carcinoma cases. TOX4 is the least understood PP1C subunit. Here we show that chromatin occupancy pattern of TOX4 resembles that of RNA polymerase II (Pol II), and its loss increases the phosphorylation of the C-terminal domain and decreases the global occupancy of Pol II. Mechanistically, it promotes promoter-proximal pause of Pol II by facilitating the recruitment of DSIF and NELF likely in a dephosphorylation dependent manner. Moreover, quantitative proteomic analyses revealed that TOX4 mainly facilitates protein dephosphorylation by PP1 phosphatases. Furthermore, Tox4 knockout in mice impairs T cell development by dysregulating genes critical for survival, proliferation and fate determination. These results lay the foundation for further understanding roles of TOX4 in transcription, development and diseases.

Double-Sided Incremental Forming (DSIF) is a rapid-prototyping manufacturing process for metal forming that, for low-volume production, is competitively energy-efficient. However, controlling the DSIF process in terms of accuracy and formability is an ongoing challenge. These control challenges arise due to a lack of understanding of the underlying deformation mechanisms in DSIF, which finite element simulations can help to unravel. However, DSIF pushes the limits of modern finite element formulations due to true strains that approach one, finite rotations, nonlinear contact, and triaxial stress states that range across multiple length scales. To confidently develop a finite element model of DSIF, an extensive verification process must be considered, which is the objective of this study. In this work, different finite element types and varying amounts of artificial acceleration are investigated, and recommendations based on efficiency and accuracy are summarized. A simplified, axisymmetric geometry was considered to reduce simulation time. For this geometry, accelerating the explicit finite element simulation by a mass factor of 105 or greater affected the stress triaxiality in the sheet by as much as 40% in some locations with respect to the quasi-static case. Additionally, the ratio of the kinetic energy to internal energy of the sheet was not a reliable indicator of whether a DSIF simulation is approximately quasi-static.

SUMMARYHistone chaperones are critical for controlling chromatin integrity during transcription, DNA replication, and DNA repair. We have discovered that the physical interaction between two essential histone chaperones, Spt6 and Spn1/Iws1, is required for transcriptional accuracy and nucleosome organization. To understand this requirement, we have isolated suppressors of anspt6mutation that disrupts the Spt6-Spn1 interaction. Several suppressors are in a third essential histone chaperone, FACT, while another suppressor is in the transcription elongation factor Spt5/DSIF. The FACT suppressors weaken FACT-nucleosome interactions and bypass the requirement for Spn1, possibly by restoring a necessary balance between Spt6 and FACT on chromatin. In contrast, the Spt5 suppressor modulates Spt6 function in a Spn1-dependent manner. Despite these distinct mechanisms, both suppressors alleviate the nucleosome organization defects caused by disruption of the Spt6-Spn1 interaction. Taken together, we have uncovered a network in which histone chaperones and other elongation factors coordinate transcriptional integrity and chromatin structure.

The transcription elongation factor Spt5 is conserved from bacteria to humans. In eukaryotes, Spt5 forms a complex with Spt4 and regulates processive transcription elongation. Recent studies on transcription elongation suggest different mechanistic roles in yeast versus mammals. Higher eukaryotes utilize Spt4-Spt5 (DSIF) to regulate promoter-proximal pausing, a transcription-regulatory mechanism that connects initiation to productive elongation. DSIF is a versatile transcription factor and has been implicated in both gene-specific regulation and transcription through nucleosomes. Future studies will further elucidate the role of DSIF in transcriptional dynamics and disentangle its inhibitory and enhancing activities in transcription.

SUMMARYGene expression is tightly controlled by Cyclin-dependent kinases (CDKs) which regulate the RNA Polymerase II (RNAPII) transcription cycle at discrete checkpoints. RNAPII pausing is a CDK9-controlled rate-limiting process that occurs shortly after initiation and is required for spatio-temporal control of transcription in multicellular organisms. We discovered that CDK9-mediated RNAPII pause-release is functionally opposed by a protein phosphatase 2A (PP2A) complex. PP2A dynamically competes for key CDK9 substrates, DSIF and RNAPII, and is recruited to transcription pausing sites by INTS6, a subunit of the Integrator complex. INTS6 depletion disrupts the Integrator-PP2A association and confers resistance to CDK9 inhibition. This results in unrestrained activity of CDK9 and dysregulation of acute transcriptional responses. Pharmacological PP2A activation amplifies RNAPII pausing mediated by CDK9 inhibitors and synergizes therapeutically in a model of MLL-rearranged leukemia. These data demonstrate that finely-tuned gene expression relies on the delicate balance of kinase and phosphatase activity throughout the transcription cycle.HIGHLIGHTSLoss of INTS6 confers resistance to CDK9 inhibitionINTS6 recruits PP2A to Integrator and chromatinPP2A/INTS6 complexes functionally oppose CDK9PP2A/INTS6 fine-tune acute transcriptional responsesSynergistic anti-cancer activity between PP2A activators and CDK9 inhibitors

Transcription by RNA polymerase II (Pol II) is carried out by an elongation complex. We previously reported an activated porcine Pol II elongation complex, EC*, encompassing the human elongation factors DSIF, PAF1 complex (PAF) and SPT6. Here we report the cryo-EM structure of the complete EC* that contains RTF1, a dissociable PAF subunit critical for chromatin transcription. The RTF1 Plus3 domain associates with Pol II subunit RPB12 and the phosphorylated C-terminal region of DSIF subunit SPT5. RTF1 also forms four α-helices that extend from the Plus3 domain along the Pol II protrusion and RPB10 to the polymerase funnel. The C-terminal ‘fastener’ helix retains PAF and is followed by a ‘latch’ that reaches the end of the bridge helix, a flexible element of the Pol II active site. RTF1 strongly stimulates Pol II elongation, and this requires the latch, possibly suggesting that RTF1 activates transcription allosterically by influencing Pol II translocation. Cryo-EM elucidation of a fully reconstituted Pol II–DSF–PAF1–SPT6 elongation complex defines the position of PAF1 subunit RTF1 and reveals contacts with the Pol II bridge helix that may allosterically stimulate transcription elongation.

Double-Sided Incremental forming (DSIF) is a flexible sheet forming method with increasing research interest in the last decade. It offers improved formability and accuracy over the conventional single point incremental forming (SPIF) although it accompanies with the possibility of tools losing contact during the forming process. Mounting a pneumatic supporting tool is an efficient solution. However, the tools may squeeze and over-bend the material when forming for a high wall angle. A possible reason may be due to inaccurate prediction of material thinning or the effect of tool deflection. This work uses a modified finite element (FE) model with dedicated toolpath design incorporated with simplified stiffness of tools and machine in order to reproduce the effect of system elasticity to the DSIF process with hydraulic supporting tool and evaluate the root causes of the over-bending problem. The numerical results are compared with experimentally produced components, focusing on the thickness evaluation and development of the tool deflection throughout the process. This comparison demonstrates the effect of the tool deflection upon the formed parts, including the geometric error and excessive deformation on the wall region. The conclusion suggests a need for a compensation based approach in the design of DISF toolpath and machine systems.

RNA polymerase II (Pol II) transcribes all protein-coding genes and many noncoding RNAs in eukaryotic genomes. Although Pol II is a complex, 12-subunit enzyme, it lacks the ability to initiate transcription and cannot consistently transcribe through long DNA sequences. To execute these essential functions, an array of proteins and protein complexes interact with Pol II to regulate its activity. In this review, we detail the structure and mechanism of over a dozen factors that govern Pol II initiation (e.g., TFIID, TFIIH, and Mediator), pausing, and elongation (e.g., DSIF, NELF, PAF, and P-TEFb). The structural basis for Pol II transcription regulation has advanced rapidly in the past decade, largely due to technological innovations in cryoelectron microscopy. Here, we summarize a wealth of structural and functional data that have enabled a deeper understanding of Pol II transcription mechanisms; we also highlight mechanistic questions that remain unanswered or controversial.

Cyclin-dependent kinase 9 (CDK9) is critical for RNA Polymerase II (Pol II) transcription initiation, elongation, and termination in several key biological processes including development, differentiation, and cell fate responses. A broad range of diseases are characterized by CDK9 malfunction, illustrating its importance in maintaining transcriptional homeostasis in basal- and signal-regulated conditions. Here we provide a historical recount of CDK9 discovery and the current models suggesting CDK9 is a central hub necessary for proper execution of different steps in the transcription cycle. Finally, we discuss the current therapeutic strategies to treat CDK9 malfunction in several disease states. Abbreviations: CDK: Cyclin-dependent kinase; Pol II: RNA Polymerase II; PIC: Pre-initiation Complex; TFIIH: Transcription Factor-II H; snoRNA: small nucleolar RNA; CycT: CyclinT1/T2; P-TEFb: Positive Transcription Elongation Factor Complex; snRNP: small nuclear ribonucleo-protein; HEXIM: Hexamethylene Bis-acetamide-inducible Protein 1/2; LARP7: La-related Protein 7; MePCE: Methylphosphate Capping Enzyme; HIV: human immunodeficiency virus; TAT: trans-activator of transcription; TAR: Trans-activation response element; Hsp70: Heat Shock Protein 70; Hsp90/Cdc37: Hsp90- Hsp90 co-chaperone Cdc37; DSIF: DRB Sensitivity Inducing Factor; NELF: Negative Elongation Factor; CPSF: cleavage and polyadenylation-specific factor; CSTF: cleavage-stimulatory factor; eRNA: enhancer RNA; BRD4: Bromodomain-containing protein 4; JMJD6: Jumonji C-domain-containing protein 6; SEC: Super Elongation Complex; ELL: eleven-nineteen Lys-rich leukemia; ENL: eleven-nineteen leukemia; MLL: mixed lineage leukemia; BEC: BRD4-containing Elongation Complex; SEC-L2/L3: SEC-like complexes; KAP1: Kruppel-associated box-protein 1; KEC: KAP1-7SK Elongation Complex; DRB: Dichloro-1-ß-D-Ribofuranosylbenzimidazole; H2Bub1: H2B mono-ubiquitination; KM: KM05382; PP1: Protein Phosphatase 1; CDK9i: CDK9 inhibitor; SHAPE: Selective 2'-hydroxyl acylation analyzed by primer extension; TE: Typical enhancer; SE : Super enhancer.

A useful method for studying the function of the mammalian RNA polymerase II takes advantage of the extreme sensitivity of its largest subunit, Rpb1, to alpha-amanitin (a-amanitin). Mutations of interest are introduced into an a-amanitin-resistant version of Rpb1, which is then expressed ectopically in cells. The phenotypes of these cells are then examined after inhibiting the endogenous wild-type polymerase with a-amanitin. Here, we show that cells that are enabled to grow in a-amanitin by expression of an a-amanitin-resistant Rpb1 exhibit changes in cell physiology that can lead to misleading experimental outcomes. The changes we have characterized include the accelerated degradation of some proteins, such as DSIF160, and the reduced rate of synthesis of others. In one series of experiments, we examined an a-amanitin-resistant construct, with a mutant C-terminal domain (CTD), that was unable to direct poly(A)-dependent transcription termination in cells growing in a-amanitin. The potential interpretation that the termination defect in this construct is due to the mutation in the CTD was rejected when the construct was found to be termination-competent in cells grown in the absence of a-amanitin. Instead, it appears that certain termination factors become limiting when the cells are grown in a-amanitin, presumably due to the a-amanitin-induced degradation we have characterized and/or to the inadequate transcription of certain genes by the a-amanitin-resistant Rpb1-containing polymerase.

The utilization of a supporting tool in Double-Sided Incremental Forming (DSIF) imposes a stabilizing compressive stress through the sheet’s thickness, increasing, thereby, the material’s formability and fatigue life. However, these favorable effects strongly depend on a steady tool-metal contact condition. This work presents a general DSIF control scheme, which augments the conventional position servo-loop with explicit force feedback control. The algorithm is examined for its robustness and effectiveness using complex geometries with varying curvatures and wall angles. The resulting parts have demonstrated enhanced material formability and geometric accuracy.