Termination Mechanism Research Articles

Recrystallization and spheroidization mechanisms in Ti6246 alloy under thermomechanical treatment

This paper provides a detailed analysis of the dynamic microstructure evolution of Ti-6Al-2Sn-4Zr-6Mo alloy (Ti6246) through a 4-pass hot rolling process. Additionally, it examines the effect of heat treatment on microstructure evolution during the post-annealing of the hot-rolled alloy. During the rolling process, dynamic recrystallization (DRX) predominantly takes place in the α-phase, leading to grain refinement and spheroidization as a consequence of DRX and lamellae spheroidization. Spheroidization of the lamellar structure is more pronounced in the 2-pass rolling process, while the level of DRX is greater in the 4-pass rolling process. The dislocations present within the α lamellae accumulate and entangle at the sub-grain boundary, leading to the formation of a high-angle grain boundary (HAGB). Meanwhile, the β phase infiltrates the grain boundaries of the α phase, leading to the formation of spheroidized equiaxed α grains. Schmid factors associated with the pyramidal slip systems are relatively high, facilitating the activation of slip systems during hot rolling processes. The evolution of the α-phase morphology during heat treatment processes in various rolling passes is primarily driven by static recrystallization (SRX) and grain boundary terminal migration mechanisms. The deformed microstructure volume fraction diminishes and transforms into substructures and recrystallized grains through post-annealing. Post-annealing facilitates the dissipation of stored deformation energy with increasing temperature, leading to a decrease in dislocation density and an enhancement in the extent of recrystallization. Accumulated deformation during rolling and the temperature of the heat treatment both play a role in affecting the static spheroidization process. Greater accumulated deformation and elevated post-annealing temperatures are advantageous for achieving grain spheroidization. The grain boundary splitting process is more pronounced, and the deformation energy is elevated during the 3,4-pass rolling.

DNA-directed termination of mammalian RNA polymerase II.

The best-studied mechanism of eukaryotic RNA polymerase II (RNAPII) transcriptional termination involves polyadenylation site-directed cleavage of the nascent RNA. The RNAPII-associated cleavage product is then degraded by XRN2, dislodging RNAPII from the DNA template. In contrast, prokaryotic RNAP and eukaryotic RNAPIII often terminate directly at T-tracts in the coding DNA strand. Here, we demonstrate a similar and omnipresent capability for mammalian RNAPII. Importantly, this termination mechanism does not require upstream RNA cleavage. Accordingly, T-tract-dependent termination can take place when XRN2 cannot be engaged. We show that T-tracts can terminate snRNA transcription independently of RNA cleavage by the Integrator complex. Importantly, we found genome-wide termination at T-tracts in promoter-proximal regions but not within protein-coding gene bodies. XRN2-dependent termination dominates downstream from protein-coding genes, but the T-tract process is sometimes used. Overall, we demonstrate global DNA-directed attrition of RNAPII transcription, suggesting that RNAPs retain the potential to terminate over T-rich sequences throughout evolution.

Mechanism of polyadenylation-independent RNA polymerase II termination.

The mechanisms underlying the initiation and elongation of RNA polymerase II (Pol II) transcription are well-studied, whereas termination remains poorly understood. Here we analyze the mechanism of polyadenylation-independent Pol II termination mediated by the yeast Sen1 helicase. Cryo-electron microscopy structures of two pretermination intermediates show that Sen1 binds to Pol II and uses its adenosine triphosphatase activity to pull on exiting RNA in the 5' direction. This is predicted to push Pol II forward, induce an unstable hypertranslocated state and destabilize the transcription bubble, thereby facilitating termination. This mechanism of transcription termination may be widely used because it is conceptually conserved in the bacterial transcription system.

Some aspects of termination of civil service in Ukraine and in the countries of the European Union

The article is devoted to the study of the peculiarities of termination of civil service in Ukraine and in some European countries. Within the framework of the article the legal grounds for termination of civil service and the procedures for the termination of administrative-service relations is analyzed. The author tries to make a partial comparative analysis of the mechanisms of termination of civil service in Ukraine and some European countries. The author put an emphasis on the need to clearly define the status of a civil servant upon termination of civil service and the expediency of implementing an effective mechanism for his legal protection upon termination of official relations on initiative of the subject of appointment, on the one hand, and of increasing the personal responsibility of civil servants for fulfilling their obligations upon termination of civil service on of their own accord, on the other hand. Understanding the concept of termination of civil service and legal grounds of such termination according to Ukrainian legislation and to legislation of foreign countries, in particular Lithuania, Czech Republic, Germany and Poland were analyzed. An intermediate conclusion that the establishing an exhaustive list of legal grounds for termination of civil service is a positive factor which is inherent in the legislation of most European countries, that prevents an abuse and arbitrary behavior by the subject of appointment and minimizes the influence of political factors on the civil service corps and guarantees its stability and perpetuity, was made. Based on the results of the analysis, the features of the realization of procedures of the termination of civil service, the notice periods for termination of civil service, both from the subject of appointment and from the civil servant, were highlighted. The author put an emphasis on the peculiarities of the termination of civil service under the martial law of Ukraine. It was concluded that the legislation of Ukraine on the termination of service relations fully complies with European standards and contributes to the effective functioning of the civil service institute in the country. That is why the opinion regarding the availability of effective and high-quality functioning of the civil service in the conditions of the European integration processes of our country, evidence of which is the existing mechanisms for protecting the rights and freedoms of civil servants before, during and after the termination of service relations and the ensuring public interests through maximum use of their potential, was expressed by the author.

Adaptive Differential Evolution with the Stagnation Termination Mechanism

Adaptive Differential Evolution with the Stagnation Termination Mechanism

Mechanisms underlying the spontaneous termination of torsades de pointes in an experimental model of long QT syndrome

BackgroundTorsades de pointes (TdP) represent a complex polymorphic ventricular tachycardia. While the triggering mechanisms of early afterdepolarization and increased dispersion of repolarization are well investigated, the sudden self-limiting termination remains poorly understood. ObjectiveThe purpose of this study was to perform analysis of TdP to investigate factors causing spontaneous termination. MethodsWe used a large data set from Langendorff experiments in isolated rabbit hearts in which drug-induced QT prolongation, bradycardia, and hypokalemia provoke TdP. We included 427 episodes with typical TdP characteristics of polymorphic self-terminating beats and twisting QRS complexes occurring in the presence of abnormal QT prolongation due to various different QT-prolonging drugs. The use of 8 monophasic action potential catheters allowed the characterization of action potential duration, configuration, and dispersion of repolarization beyond the capabilities of the surface electrocardiogram. To identify possible mechanisms of arrhythmia termination, the initial, midpoint, and terminal 3 ventricular complexes were analyzed for each episode. ResultsAn abrupt decrease in spatial dispersion over the course of a TdP episode was identified as a precursor for termination of TdP. Within the last 3 beats, a sudden significant decrease in the dispersion of repolarization was observed as a predictor of termination. In parallel, there was a decrease in action potential duration (action potential duration at 90% repolarization) before termination. Also, a change in action potential configuration (action potential duration at 90% repolarization/action potential duration at 50% repolarization ratio) in terms of the loss of action potential dome with a restitution of action potential triangulation was observed. ConclusionIn >400 TdP episodes, homogenization of myocardial repolarization with the recovery of an action potential configuration determines the termination of TdP episodes.

From efficiency to equity: A multi-user paradigm in mobile route optimization

This study addresses the challenge of optimizing vehicle mobility in urban environments, which is significant for the advancement of smart city initiatives and spatial data analysis. We introduce a novel mobile recommendation system designed for multi-user scenarios, aiming to achieve a balance between effectiveness and fairness. The system prioritizes maximizing the profitability of vehicle service providers while ensuring an equitable distribution of recommended routes among users. Our approach features a redefined objective function that integrates a fairness criterion alongside path quality optimization. We further propose PSA-DLMA (Parallel Simulated Annealing with Deep Learning-Guided Move Adaptation), a stochastic path search method that leverages deep learning to guide move and strategy selection, alongside a dynamic termination mechanism and a parallel processing strategy. We validate our methodology using recent yellow taxi data from New York City and its surroundings, conducting comprehensive experiments to assess the performance of the system. The results demonstrate the superiority of PSA-DLMA over existing state-of-the-art solutions, offering significant contributions to improving urban vehicle mobility within the smart city framework.

Growth Mechanism of Carbon Nanotubes Revealed by in situ Transmission Electron Microscopy.

Elucidating the growth mechanism of carbon nanotubes (CNTs) is critical to obtaining CNTs with desired structures and tailored properties for their practical applications. With atomic resolution imaging, in situ transmission electron microscopy (TEM) has been a key technique to reveal the microstructure and dynamics of CNTs in real time. In this review, recent advances in the development of in situ TEM with different types of environmental reactors will be introduced. The catalytic growth mechanisms of CNTs revealed by in situ TEM under realistic conditions are discussed from fundamental thermodynamics and kinetics to the detailed nucleation, growth, and termination mechanisms, including the state and phase of active catalysts, interfacial connections between catalyst and growing CNTs, and catalyst-related growth kinetics of CNTs. Great progresses have been made on how a CNT nucleates, grows and terminates, focusing on the interface dynamics and kinetic fluctuations. Finally, challenges and future directions for understanding the atomic dynamics under the real growth conditions are proposed. It is expected that breakthroughs in the fundamental growth mechanisms will pave the way to the ultimate goal of designing and controlling the atomic structures of CNTs for their applications in various devices.

Dispersion, solid solution, and covalent bond coupled to strengthen K4169/TiAl composite brazed joints: First-principles and experimental perspective

Ni/TiAl composite brazed joints could significantly reduce the aircraft's weight. However, low interfacial adhesion, coarse and brittle-hard intermetallic compounds (IMCs) seriously limited the application of Ni/TiAl composite joints in the next generation of aerospace applications. So enhanced K4169/TiAl composite joints were investigated by vacuum brazed with (Ni53.33Cr20B16.67Si10/Zr25Ti18.75Ta12.5Ni25Cu18.75) composite filler metal (CFM) designed based on cluster-plus-glue-atom model. The shear strength of the joint reached 485 MPa, comparable to the 491 MPa of TiAl substrate. The flat and brittle-hard diffusion reaction layer between Zones I and II was eliminated, simultaneously generating CrB4 dispersion strengthening due to the CFM developed with the interfacial solid-liquid space-time hysteresis effect. In Zones II and III, IMCs all transformed into Niss(Cr,Fe)[0–88], Niss(Ti, Al)[004], and Niss(Zr,Si)[11–2] of circular and oval shapes through isothermal solidification. Meanwhile, the residual stresses and hardness were distributed in reticulated cladding characteristics. Thereby, lattice distortion led to solid solution strengthening and increased plastic toughness through crack termination and bridging mechanisms, which inhibited dislocations from plugging and crack propagation. Various interfaces in Zone Ⅳ were regulated into semi- and coherent interfaces. Ni3(Ti,Al)/(Ni,Ti,Al) and (Ni,Ti,Al)/AlNi2Ti were composed of higher interfacial bonding energy (2.771 J/m2, 2.547 J/m2) and Ni-Ni covalent bonds. Interfacial covalent bonding and large interfacial bonding energy coupling strengthened Zone IV. Consequently, cracks initiated at the (Ni,Ti,Al)[013]/Ti3Al[010] and expanded rapidly into TiAl substrate. Therefore, applying this method to design CFMs and regulate the phase, grain morphology, and interface's fine structure could provide new pathways for dissimilar hard-to-join metals.

Structural basis of eukaryotic transcription termination by the Rat1 exonuclease complex

The 5´–3´ exoribonuclease Rat1/Xrn2 is responsible for the termination of eukaryotic mRNA transcription by RNAPII. Rat1 forms a complex with its partner proteins, Rai1 and Rtt103, and acts as a “torpedo” to bind transcribing RNAPII and dissociate DNA/RNA from it. Here we report the cryo-electron microscopy structures of the Rat1-Rai1-Rtt103 complex and three Rat1-Rai1-associated RNAPII complexes (type-1, type-1b, and type-2) from the yeast, Komagataella phaffii. The Rat1-Rai1-Rtt103 structure revealed that Rat1 and Rai1 form a heterotetramer with a single Rtt103 bound between two Rai1 molecules. In the type-1 complex, Rat1-Rai1 forms a heterodimer and binds to the RNA exit site of RNAPII to extract RNA into the Rat1 exonuclease active site. This interaction changes the RNA path in favor of termination (the “pre-termination” state). The type-1b and type-2 complexes have no bound DNA/RNA, likely representing the “post-termination” states. These structures illustrate the termination mechanism of eukaryotic mRNA transcription.

In Vitro and In Vivo Assessments of Newly Isolated N4-like Bacteriophage against ST45 K62 Capsular-Type Carbapenem-Resistant Klebsiella pneumoniae: vB_kpnP_KPYAP-1.

The rise of carbapenem-resistant Klebsiella pneumoniae (CRKP) presents a significant global challenge in clinical and healthcare settings, severely limiting treatment options. This study aimed to utilize a bacteriophage as an alternative therapy against carbapenem-resistant K. pneumoniae. A novel lytic N4-like Klebsiella phage, vB_kpnP_KPYAP-1 (KPYAP-1), was isolated from sewage. It demonstrated efficacy against the K62 serotype polysaccharide capsule of blaOXA-48-producing K. pneumoniae. KPYAP-1 forms small, clear plaques, has a latent period of 20 min, and reaches a growth plateau at 35 min, with a burst size of 473 plaque-forming units (PFUs) per infected cell. Phylogenetic analysis places KPYAP-1 in the Schitoviridae family, Enquatrovirinae subfamily, and Kaypoctavirus genus. KPYAP-1 employs an N4-like direct terminal repeat mechanism for genome packaging and encodes a large virion-encapsulated RNA polymerase. It lacks integrase or repressor genes, antibiotic resistance genes, bacterial virulence factors, and toxins, ensuring its safety for therapeutic use. Comparative genome analysis revealed that the KPYAP-1 genome is most similar to the KP8 genome, yet differs in tail fiber protein, indicating variations in host recognition. In a zebrafish infection model, KPYAP-1 significantly improved the survival rate of infected fish by 92% at a multiplicity of infection (MOI) of 10, demonstrating its potential for in vivo treatment. These results highlight KPYAP-1 as a promising candidate for developing phage-based therapies targeting carbapenemase-producing K. pneumoniae.

Design and Simulation of an Innovative Modular Four-Bar Linkage Robotic Arm: Analysis and Applications

In response to the issues of traditional manipulators, such as difficulty in controlling the terminal posture during motion, low control precision, and complex structure, a modular mechanical arm based on a parallel four-bar terminal posture maintenance mechanism has been designed to meet the special requirements of terminal posture maintenance. By analyzing the motion mechanism of the manipulator, the shape space of the manipulator, the terminal position space, and the relationships of forward and inverse kinematics have been derived. A simulation model of the linkage manipulator was established in the Robotic Toolbook for Matlab environment, and structural simulation analysis and kinematic equation verification were conducted. The end positioning and attitude control of different manipulators are simulated by Matlab programming. The working space of the manipulators is calculated by Monte Carlo method, and compared with that of traditional planar manipulators. The results show the validity of the kinematics equation and the rationality of the end positioning control scheme of the linkage manipulator. Finally, the experiment verifies that the attitude retention rate of the modular decoupling manipulator is 97.78% under high load, which verifies the reliability of this scheme. In addition, the manipulator can simplify the complexity of the mechanical structure and complete the control requirements of the terminal attitude under the premise of increasing the working space as much as possible, which lays a foundation for the design and optimization of the manipulator in the field of modular terminal attitude.

Studies on the Termination Behavior of Styrenic Free‐Radicals

AbstractIn order to provide more evidence to understand the termination mechanism in styrene free‐radical polymerization, (1‐bromoethyl) benzene and polystyrene with bromide end group (PSt‐Br) synthesized by atom transfer radical polymerization (ATRP) were used as the model compound and model polymer to study the termination behavior of styrenic radical by atom transfer radical coupling (ATRC), and then the NMR chemical shifts of the head‐to‐head enchainment from coupling termination were obtained. SEC and different NMR measurement methods were used to analyze and characterize the products. It is confirmed that there is no bimolecular disproportionation termination between styrenic free radicals, but only coupling termination to form the head‐to‐head enchainment. The proton and carbon chemical shifts of the head‐to‐head enchainment from coupling termination for (1‐bromoethyl) benzene are 2.7–2.86 ppm and ~47.3 ppm. As for the polystyrene through ATRC from PSt‐Br, the corresponding chemical shifts are 2.61–3.29 ppm and ~46.4 ppm, respectively, they are almost the same as the data in our previous paper (~3.05 ppm, ~46.6 ppm) for polystyrene initiated by BPO, proving that the chain termination in free‐radical polymerization of styrene initiated by BPO occurs through primary termination between chain radical and primary radical rather than bimolecular termination between chain radicals.

Silicon Nitride Anodes for Li-Ion Batteries: Understanding the Origin of Enhanced Cycle Stability over Silicon with Operando AFM

Silicon is highly coveted as the potential anode for next generation batteries, owing to its high specific capacity (3579 mAh g-1, Li3.75Si), high abundance and low cost. However, the large volume expansion of silicon during lithiation (up to 300 %) poses inherent challenges, namely cracking/fracturing of the anode which constitutes to drastic capacity fade, thereby greatly limiting the long-term cyclability and ultimately leads to terminal cell failure mechanisms such as delamination [1]. A promising alternative is the use of conversion alloying materials such as silicon nitride (SiNx). During the initial lithiation (first cycle), these materials irreversibly convert into a mixture of matrix (LixSiyNz) and the redox active electrode (LixSi). In subsequent cycles, the improved cycle performance and lifetime is attributed to the matrix helping buffer the volume expansion of the Si particles, thus mitigating against the aforementioned degradation pathways and ultimately extending the cycle performance lifetime. Currently, insights into the silicon nitride alloying conversion reaction are limited. There remains debate in the literature around whether the matrix is redox active, and uncertainty over the topographical distribution of the anode, specifically whether discrete domains of silicon and matrix are generated in situ, or the silicon nitride remains as amorphous solid solution of silicon and lithiated nitridosilicates, where only the short-range chemical environment resembles a given phase [2-4].At time of writing there have been no operando observations of the reaction. In this presentation, we address this by studying pulse laser deposited (PLD) SiNx thin films with operando electrochemical atomic force microscopy (EC-AFM). This technique (performed in a glovebox under inert Ar conditions) enables insights into the morphological, mechanical, chemical, and physical properties of battery materials when they evolve under electrochemical control. Crucially, operando measurements enable real time detection of the morphological changes of the SiNx anode during the formation cycle, in conjunction with Young’s Modulus mapping enabling phase-identification of the matrix and silicon components. First, we report thorough electrochemical characterisation of SiNx films with varying composition (x), demonstrating performance equivalent to the best-in-class. Then, EC-AFM studies are communicated; comparisons are made between SiNx and Si films to elucidate the alloying conversion reaction, how it impacts the morphological evolution of the anode, and consequently, establish the origins of the improved SiNx cyclability. References Shi, F., Song, Z., Ross, P.N., Somorjai, G.A., Ritchie, R.O. and Komvopoulos, K., 2016. Failure mechanisms of single-crystal silicon electrodes in lithium-ion batteries. Nature communications, 7(1), p.11886.Ulvestad, A., Mæhlen, J.P. and Kirkengen, M., 2018. Silicon nitride as anode material for Li-ion batteries: Understanding the SiNx conversion reaction. Journal of Power Sources, 399, pp.414-421.Ulvestad, A., Skare, M.O., Foss, C.E., Krogsæter, H., Reichstein, J.F., Preston, T.J., Mæhlen, J.P., Andersen, H.F. and Koposov, A.Y., 2021. Stoichiometry-controlled reversible lithiation capacity in nanostructured silicon nitrides enabled by in situ conversion reaction. ACS nano, 15(10), pp.16777-16787.Kilian, S.O., Wankmiller, B., Sybrecht, A.M., Twellmann, J., Hansen, M.R. and Wiggers, H., 2022. Active Buffer Matrix in Nanoparticle‐Based Silicon‐Rich Silicon Nitride Anodes Enables High Stability and Fast Charging of Lithium‐Ion Batteries. Advanced Materials Interfaces, 9(26), p.2201389.

Single-molecule reconstruction of eukaryotic factor-dependent transcription termination

Factor-dependent termination uses molecular motors to remodel transcription machineries, but the associated mechanisms, especially in eukaryotes, are poorly understood. Here we use single-molecule fluorescence assays to characterize in real time the composition and the catalytic states of Saccharomyces cerevisiae transcription termination complexes remodeled by Sen1 helicase. We confirm that Sen1 takes the RNA transcript as its substrate and translocates along it by hydrolyzing multiple ATPs to form an intermediate with a stalled RNA polymerase II (Pol II) transcription elongation complex (TEC). We show that this intermediate dissociates upon hydrolysis of a single ATP leading to dissociation of Sen1 and RNA, after which Sen1 remains bound to the RNA. We find that Pol II ends up in a variety of states: dissociating from the DNA substrate, which is facilitated by transcription bubble rewinding, being retained to the DNA substrate, or diffusing along the DNA substrate. Our results provide a complete quantitative framework for understanding the mechanism of Sen1-dependent transcription termination in eukaryotes.

Particle Swarm Optimization Algorithm Using Velocity Pausing and Adaptive Strategy

Particle swarm optimization (PSO) as a swarm intelligence-based optimization algorithm has been widely applied to solve various real-world optimization problems. However, traditional PSO algorithms encounter issues such as premature convergence and an imbalance between global exploration and local exploitation capabilities when dealing with complex optimization tasks. To address these shortcomings, an enhanced PSO algorithm incorporating velocity pausing and adaptive strategies is proposed. By leveraging the search characteristics of velocity pausing and the terminal replacement mechanism, the problem of premature convergence inherent in standard PSO algorithms is mitigated. The algorithm further refines and controls the search space of the particle swarm through time-varying inertia coefficients, symmetric cooperative swarms concepts, and adaptive strategies, balancing global search and local exploitation. The performance of VASPSO was validated on 29 standard functions from Cec2017, comparing it against five PSO variants and seven swarm intelligence algorithms. Experimental results demonstrate that VASPSO exhibits considerable competitiveness when compared with 12 algorithms. The relevant code can be found on our project homepage.

CLK2 mediates IκBα-independent early termination of NF-κB activation by inducing cytoplasmic redistribution and degradation

Activation of the NF-κB pathway is strictly regulated to prevent excessive inflammatory and immune responses. In a well-known negative feedback model, IκBα-dependent NF-κB termination is a delayed response pattern in the later stage of activation, and the mechanisms mediating the rapid termination of active NF-κB remain unclear. Here, we showed IκBα-independent rapid termination of nuclear NF-κB mediated by CLK2, which negatively regulated active NF-κB by phosphorylating the RelA/p65 subunit of NF-κB at Ser180 in the nucleus to limit its transcriptional activation through degradation and nuclear export. Depletion of CLK2 increased the production of inflammatory cytokines, reduced viral replication and increased the survival of the mice. Mechanistically, CLK2 phosphorylated RelA/p65 at Ser180 in the nucleus, leading to ubiquitin‒proteasome-mediated degradation and cytoplasmic redistribution. Importantly, a CLK2 inhibitor promoted cytokine production, reduced viral replication, and accelerated murine psoriasis. This study revealed an IκBα-independent mechanism of early-stage termination of NF-κB in which phosphorylated Ser180 RelA/p65 turned off posttranslational modifications associated with transcriptional activation, ultimately resulting in the degradation and nuclear export of RelA/p65 to inhibit excessive inflammatory activation. Our findings showed that the phosphorylation of RelA/p65 at Ser180 in the nucleus inhibits early-stage NF-κB activation, thereby mediating the negative regulation of NF-κB.

A widely conserved protein Rof inhibits transcription termination factor Rho and promotes Salmonella virulence program

Transcription is crucial for the expression of genetic information and its efficient and accurate termination is required for all living organisms. Rho-dependent termination could rapidly terminate unwanted premature RNAs and play important roles in bacterial adaptation to changing environments. Although Rho has been discovered for about five decades, the regulation mechanisms of Rho-dependent termination are still not fully elucidated. Here we report that Rof is a conserved antiterminator and determine the cryogenic electron microscopy structure of Rho-Rof antitermination complex. Rof binds to the open-ring Rho hexamer and inhibits the initiation of Rho-dependent termination. Rof’s N-terminal α-helix undergoes conformational changes upon binding with Rho, and is key in facilitating Rof-Rho interactions. Rof binds to Rho’s primary binding site (PBS) and excludes Rho from binding with PBS ligand RNA at the initiation step. Further in vivo analyses in Salmonella Typhimurium show that Rof is required for virulence gene expression and host cell invasion, unveiling a physiological function of Rof and transcription termination in bacterial pathogenesis.

From Ground-State to Excited-State Activation Modes: Flavin-Dependent "Ene"-Reductases Catalyzed Non-natural Radical Reactions.

Enzymes are desired catalysts for chemical synthesis, because they can be engineered to provide unparalleled levels of efficiency and selectivity. Yet, despite the astonishing array of reactions catalyzed by natural enzymes, many reactivity patterns found in small molecule catalysts have no counterpart in the living world. With a detailed understanding of the mechanisms utilized by small molecule catalysts, we can identify existing enzymes with the potential to catalyze reactions that are currently unknown in nature. Over the past eight years, our group has demonstrated that flavin-dependent "ene"-reductases (EREDs) can catalyze various radical-mediated reactions with unparalleled levels of selectivity, solving long-standing challenges in asymmetric synthesis.This Account presents our development of EREDs as general catalysts for asymmetric radical reactions. While we have developed multiple mechanisms for generating radicals within protein active sites, this account will focus on examples where flavin mononucleotide hydroquinone (FMNhq) serves as an electron transfer radical initiator. While our initial mechanistic hypotheses were rooted in electron-transfer-based radical initiation mechanisms commonly used by synthetic organic chemists, we ultimately uncovered emergent mechanisms of radical initiation that are unique to the protein active site. We will begin by covering intramolecular reactions and discussing how the protein activates the substrate for reduction by altering the redox-potential of alkyl halides and templating the charge transfer complex between the substrate and flavin-cofactor. Protein engineering has been used to modify the fundamental photophysics of these reactions, highlighting the opportunity to tune these systems further by using directed evolution. This section highlights the range of coupling partners and radical termination mechanisms available to intramolecular reactions.The next section will focus on intermolecular reactions and the role of enzyme-templated ternary charge transfer complexes among the cofactor, alkyl halide, and coupling partner in gating electron transfer to ensure that it only occurs when both substrates are bound within the protein active site. We will highlight the synthetic applications available to this activation mode, including olefin hydroalkylation, carbohydroxylation, arene functionalization, and nitronate alkylation. This section also discusses how the protein can favor mechanistic steps that are elusive in solution for the asymmetric reductive coupling of alkyl halides and nitroalkanes. We are aware of several recent EREDs-catalyzed photoenzymatic transformations from other groups. We will discuss results from these papers in the context of understanding the nuances of radical initiation with various substrates.These biocatalytic asymmetric radical reactions often complement the state-of-the-art small-molecule-catalyzed reactions, making EREDs a valuable addition to a chemist's synthetic toolbox. Moreover, the underlying principles studied with these systems are potentially operative with other cofactor-dependent proteins, opening the door to different types of enzyme-catalyzed radical reactions. We anticipate that this Account will serve as a guide and inspire broad interest in repurposing existing enzymes to access new transformations.

Compatibility of termination mechanisms in bacterial transcription with inference on eukaryotic models.

Transcription termination has evolved to proceed through diverse mechanisms. For several classes of terminators, multiple models have been debatably proposed. Recent single-molecule studies on bacterial terminators have resolved several long-standing controversies. First, termination mode or outcome is twofold rather than single. RNA is released alone before DNA or together with DNA from RNA polymerase (RNAP), i.e. with RNA release for termination, RNAP retains on or dissociates off DNA, respectively. The concomitant release, described in textbooks, results in one-step decomposition of transcription complexes, and this 'decomposing termination' prevails at ρ factor-dependent terminators. Contrastingly, the sequential release was recently discovered abundantly from RNA hairpin-dependent intrinsic terminations. RNA-only release allows RNAP to diffuse on DNA in both directions and recycle for reinitiation. This 'recycling termination' enables one-dimensional reinitiation, which would be more expeditious than three-dimensional reinitiation by RNAP dissociated at decomposing termination. Second, while both recycling and decomposing terminations occur at a hairpin-dependent terminator, four termination mechanisms compatibly operate at a ρ-dependent terminator with ρ in alternative modes and even intrinsically without ρ. RNA-bound catch-up ρ mediates recycling termination first and decomposing termination later, while RNAP-prebound stand-by ρ invokes only decomposing termination slowly. Without ρ, decomposing termination occurs slightly and sluggishly. These four mechanisms operate on distinct timescales, providing orderly fail-safes. The stand-by mechanism is benefited by terminational pause prolongation and modulated by accompanying riboswitches more greatly than the catch-up mechanisms. Conclusively, any mechanism alone is insufficient to perfect termination, and multiple mechanisms operate compatibly to achieve maximum possible efficiency under separate controls.