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1. Reaction Coordinate and Thermodynamics of Base Flipping in RNA

   https://doi.org/10.1021/acs.jctc.0c01199  

   Levintov, Lev; Paul, Sanjib; Vashisth, Harish (February 2021, Journal of Chemical Theory and Computation)

   null (Ed.)

   Base flipping is a key biophysical event involved in recognition of various ligands by ribonucleic acid (RNA) molecules. However, the mechanism of base flipping in RNA remains poorly understood, in part due to the lack of atomistic details on complex rearrangements in neighboring bases. In this work, we applied transition path sampling (TPS) methods to study base flipping in a double-stranded RNA (dsRNA) molecule that is known to interact with RNA-editing enzymes through this mechanism. We obtained an ensemble of 1000 transition trajectories to describe the base-flipping process. We used the likelihood maximization method to determine the refined reaction coordinate (RC) consisting of two collective variables (CVs), a distance and a dihedral angle between nucleotides that form stacking interactions with the flipping base. The free energy profile projected along the refined RC revealed three minima, two corresponding to the initial and final states and one for a metastable state. We suggest that the metastable state likely represents a wobbled conformation of nucleobases observed in NMR studies that is often characterized as the flipped state. The analyses of reactive trajectories further revealed that the base flipping is coupled to a global conformational change in a stem-loop of dsRNA. 

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2. Oxidation of phenylalanyl-tRNA synthetase positively regulates translational quality control

   https://doi.org/10.1073/pnas.1901634116  

   Steiner, Rebecca E.; Kyle, Amanda M.; Ibba, Michael (May 2019, Proceedings of the National Academy of Sciences)

   Accurate translation of the genetic code is maintained in part by aminoacyl-tRNA synthetases (aaRS) proofreading mechanisms that ensure correct attachment of a cognate amino acid to a transfer RNA (tRNA). During environmental stress, such as oxidative stress, demands on aaRS proofreading are altered by changes in the availability of cytoplasmic amino acids. For example, oxidative stress increases levels of cytotoxic tyrosine isomers, noncognate amino acids normally excluded from translation by the proofreading activity of phenylalanyl-tRNA synthetase (PheRS). Here we show that oxidation of PheRS induces a conformational change, generating a partially unstructured protein. This conformational change does not affect Phe or Tyr activation or the aminoacylation activity of PheRS. However, in vitro and ex vivo analyses reveal that proofreading activity to hydrolyze Tyr-tRNA Phe is increased during oxidative stress, while the cognate Phe-tRNA Phe aminoacylation activity is unchanged. In HPX − , Escherichia coli that lack reactive oxygen-scavenging enzymes and accumulate intracellular H 2 O 2 , we found that PheRS proofreading is increased by 11%, thereby providing potential protection against hazardous cytoplasmic m -Tyr accumulation. These findings show that in response to oxidative stress, PheRS proofreading is positively regulated without negative effects on the enzyme’s housekeeping activity in translation. Our findings also illustrate that while the loss of quality control and mistranslation may be beneficial under some conditions, increased proofreading provides a mechanism for the cell to appropriately respond to environmental changes during oxidative stress. 

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3. Applications of High-Pressure Technology for High-Entropy Alloys: A Review

   https://doi.org/10.3390/met9080867  

   Dong, Wanqing; Zhou, Zheng; Zhang, Mengdi; Ma, Yimo; Yu, Pengfei; Liaw, Peter K.; Li, Gong (August 2019, Metals)

   High-entropy alloys are a new type of material developed in recent years. It breaks the traditional alloy-design conventions and has many excellent properties. High-pressure treatment is an effective means to change the structures and properties of metal materials. The pressure can effectively vary the distance and interaction between molecules or atoms, so as to change the bonding mode, and form high-pressure phases. These new material states often have different structures and characteristics, compared to untreated metal materials. At present, high-pressure technology is an effective method to prepare alloys with unique properties, and there are many techniques that can achieve high pressures. The most commonly used methods include high-pressure torsion, large cavity presses and diamond-anvil-cell presses. The materials show many unique properties under high pressures which do not exist under normal conditions, providing a new approach for the in-depth study of materials. In this paper, high-pressure (HP) technologies applied to high-entropy alloys (HEAs) are reviewed, and some possible ways to develop good properties of HEAs using HP as fabrication are introduced. Moreover, the studies of HEAs under high pressures are summarized, in order to deepen the basic understanding of HEAs under high pressures, which provides the theoretical basis for the application of high-entropy alloys. 

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4. Direct sequencing of total S. cerevisiae tRNAs by LC-MS/MS

   https://doi.org/10.1261/rna.079656.123  

   Jones, Joshua D; Simcox, Kaley M; Kennedy, Robert T; Koutmou, Kristin S (May 2023, RNA)

   Among RNAs, transfer RNAs (tRNAs) contain the widest variety of abundant post-transcriptional chemical modifications. These modifications are crucial for tRNAs to participate in protein synthesis, promoting proper tRNA structure and aminoacylation, facilitating anticodon:codon recognition, and ensuring the reading frame maintenance of the ribosome. While tRNA modifications were long thought to be stoichiometric, it is becoming increasingly apparent that these modifications can change dynamically in response to the cellular environment. The ability to broadly characterize the fluctuating tRNA modification landscape will be essential for establishing the molecular level contributions of individual sites of tRNA modification. The locations of modifications within individual tRNA sequences can be mapped using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In this approach, a single tRNA species is purified, treated with ribonucleases and the resulting single-stranded RNA products are subject to LC-MS/MS analysis. The application of LC-MS/MS to study tRNAs is limited by the necessity of analyzing one tRNA at a time because the digestion of total tRNA mixtures by commercially available ribonucleases produces many short digestion products unable to be uniquely mapped back to a single site within a tRNA. We overcame these limitations by taking advantage of the highly structured nature of tRNAs to prevent the full digestion by single-stranded RNA specific ribonucleases. Folding total tRNA prior to digestion allowed us to sequence S. cerevisiae tRNAs with up to 97% sequence coverage for individual tRNA species by LC-MS/MS. This method presents a robust avenue for directly detecting the distribution of modifications in total tRNAs. 

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5. The power and pitfalls of AlphaFold2 for structure prediction beyond rigid globular proteins

   https://doi.org/10.1038/s41589-024-01638-w  

   Agarwal, Vinayak; McShan, Andrew C (June 2024, Nature Chemical Biology)

   Artificial intelligence-driven advances in protein structure prediction in recent years have raised the question: has the protein structure-prediction problem been solved? Here, with a focus on nonglobular proteins, we highlight the many strengths and potential weaknesses of DeepMind’s AlphaFold2 in the context of its biological and therapeutic applications. We summarize the subtleties associated with evaluation of AlphaFold2 model quality and reliability using the predicted local distance difference test (pLDDT) and predicted aligned error (PAE) values. We highlight various classes of proteins that AlphaFold2 can be applied to and the caveats involved. Concrete examples of how AlphaFold2 models can be integrated with experimental data in the form of small-angle X-ray scattering (SAXS), solution NMR, cryo-electron microscopy (cryo-EM) and X-ray diffraction are discussed. Finally, we highlight the need to move beyond structure prediction of rigid, static structural snapshots toward conformational ensembles and alternate biologically relevant states. The overarching theme is that careful consideration is due when using AlphaFold2-generated models to generate testable hypotheses and structural models, rather than treating predicted models as de facto ground truth structures. 

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