Search

Nucleotide selection is crucial for transcription fidelity control, in particular, for viral T7 RNA polymerase (RNAP) lack of proofreading activity. It has been recognized that multiple kinetic checkpoints exist prior to full nucleotide incorporation. In this work, we implemented intensive atomistic molecular dynamics (MD) simulations to quantify how strong the nucleotide selection is at the initial checkpoint of an elongation cycle of T7 RNAP. The incoming nucleotides bind into a preinsertion site where a critical tyrosine residue locates nearby to assist the nucleotide selection. We calculated the relative binding free energy between a noncognate nucleotide and a cognate one at a preinsertion configuration via alchemical simulations, showing that a small selection free energy or the binding free energy difference (∼3 kBT) exists between the two nucleotides. Indeed, another preinsertion configuration favored by the noncognate nucleotides was identified, which appears to be off path for further nucleotide insertion and additionally assists the nucleotide selection. By chemical master equation (CME) approach, we show that the small selection free energy at the preinsertion site along with the off-path noncognate nucleotide filtering can help substantially to reduce the error rate and to maintain the elongation rate high in the T7 RNAP transcription.

Pyrophosphate ion (PPi) release during transcription elongation is a signature step in each nucleotide addition cycle. The kinetics and energetics of the process as well as how it proceeds with substantial conformational changes of the polymerase complex determine the mechano-chemical coupling mechanism of the transcription elongation. Here we investigated detailed dynamics of the PPi release process in a single-subunit RNA polymerase (RNAP) from bacteriophage T7, implementing all-atom molecular dynamics (MD) simulations. We obtained a jump-from-cavity kinetic model of the PPi release utilizing extensive nanosecond MD simulations. We found that the PPi release in T7 RNAP is initiated by the PPi dissociation from two catalytic aspartic acids, followed by a comparatively slow jump-from-cavity activation process. Combining with a number of microsecond long MD simulations, we also found that the activation process is hindered by charged residue associations as well as by local steric and hydrogen bond interactions. On the other hand, the activation is greatly assisted by a highly flexible lysine residue Lys472 that swings its side chain to pull PPi out. The mechanism can apply in general to single subunit RNA and DNA polymerases with similar molecular structures and conserved key residues. Remarkably, the flexible lysine or arginine residue appears to be a universal module that assists the PPi release even in multi-subunit RNAPs with charge facilitated hopping mechanisms. We also noticed that the PPi release is not tightly coupled to opening motions of an O-helix on the fingers domain of T7 RNAP according to the microsecond MD simulations. Our study thus supports the Brownian ratchet scenario of the mechano-chemical coupling in the transcription elongation of the single-subunit polymerase., Author Summary RNA polymerases (RNAPs) are usually recognized to work as Brownian ratchet machines, with mechanical movements and chemical reactions loosely coupled during transcription elongation. Nevertheless, structural studies on the single-subunit T7 RNAP had suggested an alternative power stroke scenario, which requires the PPi product release reaction tightly couples with or drives the polymerase translocation. To resolve the conflicting views in the mechano-chemical coupling scenario, we conducted atomistic molecular dynamics (MD) simulations to investigate the PPi release mechanism of T7 RNAP. Using a large number of nanosecond short simulations, we constructed the Markov state model (MSM) and found that the PPi release undergoes a jump-from-cavity activation process, unlikely to further support the translocation. The MSM implementations to multi-subunit RNAPs previously demonstrated instead the charge facilitated hopping mechanisms of the PPi release. To further explore essential slow motions during the PPi release, we also performed microsecond long MD simulations. The PPi release does not appear to be tightly coupled to the rotational opening of an O-helix that is directly tied to the polymerase translocation. Hence, the study again disfavors the power stroke scenario. Remarkably, we discovered a key residue Lys472 that is able to assist PPi to jump out of the cavity. The mechanism can be general to a group of polymerases sharing conserved structural features with T7 RNAP, while the lysine or arginine module to assist the PPi release can be universal to both the single and multi-subunit RNAPs, even though the overall structural features and the PPi release mechanisms are significantly different.

Las situaciones que plantean problemas éticos en relación con la nutrición y la hidratación son muy frecuentes hoy día, principalmente a nivel hospitalario tanto en pacientes terminales como en otro tipo de pacientes que precisen de hidronutrición. En este artículo se pretende analizar las sencillas en sus fundamentos, a la luz de valores éticos ampliamente aceptados, para intentar enunciar un modo de actuación claro que ayude en su resolución a los clínicos que han de tomar estas difíciles decisiones. Las situaciones problema analizadas incluyen, entre otras, si la hidratación y la nutrición deben considerarse medidas terapéuticas o cuidados básicos, y si presentan iguales aspectos éticos la nutrición enteral y la parenteral. Conditions that pose ethical problems related to nutrition and hydration are very common nowdays, particularly within Hospitals among terminally ill patients and other patients who require nutrition and hydration. In this article we intend to analyze some circumstances, according to widely accepted ethical values, in order to outline a clear action model to help clinicians in making such difficult decisions. The problematic situations analyzed include: should hydration and nutrition be considered basic care or therapeutic measures?, and the ethical aspects of enteral versus parenteral nutrition.

Las situaciones que plantean problemas éticos en relación con la nutrición y la hidratación son muy frecuentes hoy día, principalmente a nivel hospitalario tanto en pacientes terminales como en otro tipo de pacientes que precisen de hidronutrición. En este artículo se pretende analizar las sencillas en sus fundamentos, a la luz de valores éticos ampliamente aceptados, para intentar enunciar un modo de actuación claro que ayude en su resolución a los clínicos que han de tomar estas difíciles decisiones. Las situaciones problema analizadas incluyen, entre otras, si la hidratación y la nutrición deben considerarse medidas terapéuticas o cuidados básicos, y si presentan iguales aspectos éticos la nutrición enteral y la parenteral.

Bacteriophage T7 RNA polymerase (RNAP) serves a prototypical system to study general physical mechanisms of transcription due to its relative simple structures along with self-sufficient and strong transcription activities. We computationally study the pyrophosphate ion (PPi) product release and DNA translocation processes during the T7 RNAP elongation with unprecedented structural dynamics detail. We implemented extensive all-atom molecular dynamics (MD) simulations and constructed the Markov state model (MSM). The MSM reveals a jump-from-cavity PPi release mechanism, which distinguishes from charge facilitated hopping mechanisms identified previously for bacterial and eukaryotic multi-subunit RNAPs. Furthermore, we performed steered MD simulations and microsecond long MD simulations to explore additional couplings and slow motions. We found that the PPi release does not appear to be tightly coupled to opening of an O-helix that is directly tied to the DNA translocation. Hence, the study disfavors a power stroke mechanism of the RNAP elongation. In particular, we discovered a key lysine/arginine residue that assists the PPi release and the module appears to be universal not only to structurally similar polymerases but also to the multi-subunit RNAPs, even though their overall structures and mechanisms look quite different. We also want to understand why the viral RNAP does not backtrack during its Brownian ratcheting.

It has been demonstrated that osteoarthritis (OA) is activity related and may worsen when joint contact stress becomes excessive due to overloading. Hence, joint alignment and loading are considered to be the key biomechanical determinants for OA. The initiation of pathologic changes in the knee has been described by the mechanism termed, "vicious cycle" in which joint axial malalignment creates excessive stresses to the localized joint cartilage/subchondral bone regions and the surrounding soft tissue which in turn produces more laxity and joint deformity and thus repeats the cyclic degradation mechanism. If this degenerative cycle can be broken with joint alignment surgery such as osteotomy, a procedure to realign the knee joint and thus redistribute joint forces applied to each compartment, performed properly and at the appropriate time, the osteoarthritic disease process can be decelerated and even reversed. The main goals of this paper are to emphasize the importance of accurate preoperative planning for osteotomy in order to properly correct joint alignment, and to justify the application of an existing computer program, OASIS (Osteotomy Analysis and Simulation Software) using plain radiographs to perform appropriate surgical planning. Normal subjects and knee osteotomy patients were studied to establish a database for the purpose of establishing the utility and efficacy of the presently proposed concept. We wish to rationalize knee osteotomy as a preferred and cost-effective treatment for patients with early symptoms of OA in the knee. This paper presents a new concept of preoperative planning for knee osteotomy based on the underlying etiology of the disease and biomechanical viewpoint with strong emphasis on surgical treatment rationales. The established principles in this paper can be applied to other joints of the body and will help implement preventive measures and other non-surgical means to manage patients with axial malalignment or early degenerative changes.