Optical Control of Transcription: Genetically Encoded Photoswitchable Variants of T7 RNA Polymerase

Light-sensing protein domains that link an exogenous light signal to the activity of an enzyme have attracted much attention for the engineering of new regulatory mechanisms into proteins and for studying the dynamic behavior of intracellular reactions and reaction cascades. Light–oxygen–voltage (LOV) photoreceptors are blue-light-sensing modules that have been intensely characterized for this purpose and linked to several proteins of interest. For the successful application of these tools, it is crucial to identify appropriate fusion strategies for combining sensor and enzyme domains that sustain activity and light-induced responsivity. Terminal fusion of LOV domains is the natural strategy; however, this is not transferrable to T7 RNA polymerase because both of its termini are involved in catalysis. It is shown herein that it is possible to covalently insert LOV domains into the polymerase protein, while preserving its activity and generating new light-responsive allosteric coupling.     

A Systematic Study on the Optimal Nucleotide Analogue Concentration and Rate Limiting Nucleotide of the SARS-CoV-2 RNA-Dependent RNA Polymerase

The current COVID-19 pandemic has highlighted the necessity of more efficient antiviral compounds. The antiviral efficacy of adenosine-based analogs, the main repurposed drugs for SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) inhibition, is mainly assessed through in vitro or cell-free polymerization assays, under arbitrary conditions that do not reflect the physiological environment. We show that SARS-CoV-2 RdRp inhibition efficiency of remdesivir and cordycepin, two common adenosine analogs, is influenced by endogenous adenosine level, and that the current clinically approved concentrations for COVID-19 treatment are suboptimal for effective RdRp inhibition. Furthermore, we identified GTP as the rate-limiting nucleotide of SARS-CoV-2 replication. Our results demonstrate that nucleotide sensitivity of the RdRp complex and competition of nucleoside analog drugs against endogenous concentrations of nucleotides are crucial elements to be considered when designing new SARS-CoV-2 antiviral compounds.     

State-of-the-Art Molecular Dynamics Simulation Studies of RNA-Dependent RNA Polymerase of SARS-CoV-2

Molecular dynamics (MD) simulations are powerful theoretical methods that can reveal biomolecular properties, such as structure, fluctuations, and ligand binding, at the level of atomic detail. In this review article, recent MD simulation studies on these biomolecular properties of the RNA-dependent RNA polymerase (RdRp), which is a multidomain protein, of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are presented. Although the tertiary structures of RdRps in SARS-CoV-2 and SARS-CoV are almost identical, the RNA synthesis activity of RdRp of SARS-CoV is higher than SARS-CoV-2. Recent MD simulations observed a difference in the dynamic properties of the two RdRps, which may cause activity differences. RdRp is also a drug target for Coronavirus disease 2019 (COVID-19). Nucleotide analogs, such as remdesivir and favipiravir, are considered to be taken up by RdRp and inhibit RNA replication. Recent MD simulations revealed the recognition mechanism of RdRp for these drug molecules and adenosine triphosphate (ATP). The ligand-recognition ability of RdRp decreases in the order of remdesivir, favipiravir, and ATP. As a typical recognition process, it was found that several lysine residues of RdRp transfer these ligand molecules to the binding site such as a “bucket brigade.” This finding will contribute to understanding the mechanism of the efficient ligand recognition by RdRp. In addition, various simulation studies on the complexes of SARS-CoV-2 RdRp with several nucleotide analogs are reviewed, and the molecular mechanisms by which these compounds inhibit the function of RdRp are discussed. The simulation studies presented in this review will provide useful insights into how nucleotide analogs are recognized by RdRp and inhibit the RNA replication.     

Structural Study of the Partially Disordered Full‐Length δ Subunit of RNA Polymerase from Bacillus subtilis

The partially disordered δ subunit of RNA polymerase was studied by various NMR techniques. The structure of the well‐folded N‐terminal domain was determined based on inter‐proton distances in NOESY spectra. The obtained structural model was compared to the previously determined structure of a truncated construct (lacking the C‐terminal domain). Only marginal differences were identified, thus indicating that the first structural model was not significantly compromised by the absence of the C‐terminal domain. Various ¹⁵N relaxation experiments were employed to describe the flexibility of both domains. The relaxation data revealed that the C‐terminal domain is more flexible, but its flexibility is not uniform. By using paramagnetic labels, transient contacts of the C‐terminal tail with the N‐terminal domain and with itself were identified. A propensity of the C‐terminal domain to form β‐type structures was obtained by chemical shift analysis. Comparison with the paramagnetic relaxation enhancement indicated a well‐balanced interplay of repulsive and attractive electrostatic interactions governing the conformational behavior of the C‐terminal domain. The results showed that the δ subunit consists of a well‐ordered N‐terminal domain and a flexible C‐terminal domain that exhibits a complex hierarchy of partial ordering.     

Molecular Dynamics Studies on the Structural Stability Prediction of SARS-CoV-2 Variants Including Multiple Mutants

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused the Coronavirus Disease (COVID-19) pandemic worldwide. The spike protein in SARS-CoV-2 fuses with and invades cells in the host respiratory system by binding to angiotensin-converting enzyme 2 (ACE2). The spike protein, however, undergoes continuous mutation from a D614G single mutant to an omicron variant, including multiple mutants. In this study, variants, including multiple mutants (double, triple mutants, B.1.620, delta, alpha, delta_E484Q, mu, and omicron) were investigated in patients. The 3D structure of the full-length spike protein was used in conformational analysis depending on the SARS-CoV-2 variants. The structural stability of the variant types was analyzed based on the distance between the receptor-binding domain (RBD) of each chain in the spike protein and the binding free energy between the spike protein and bound ACE2 in the one-, two-, and three-open-complex forms using molecular dynamics (MD) simulation. Omicron variants, the most prevalent in the recent history of the global pandemic, which consist of 32 mutations, showed higher stability in all open-complex forms compared with that of the wild type and other variants. We suggest that the conformational stability of the spike protein is the one of the important determinants for the differences in viral infectivity among variants, including multiple mutants.     

改性淀粉在环境和生化工程中的应用

介绍了改性淀粉在环境工程和生物工程中的研究和应用,重点评述了酯化淀粉、醚化淀粉、接枝共聚淀粉、复合淀粉絮凝剂、淀粉微球等改性淀粉的应用研究进展,对今后的研究方向提出了一些建议。     

The Fight against the Influenza A Virus H1N1: Synthesis,Molecular Modeling,and Biological Evaluation of Benzofurazan Derivatives as Viral RNA Polymerase Inhibitors

The influenza RNA polymerase complex, which consists of the three subunits PA, PB1, and PB2, is a promising target for the development of new antiviral drugs. A large library of benzofurazan compounds was synthesized and assayed against influenza virus A/WSN/33 (H1N1). Most of the new derivatives were found to act by inhibiting the viral RNA polymerase complex through disruption of the complex formed between subunits PA and PB1. Docking studies were also performed to elucidate the binding mode of benzofurazans within the PB1 binding site in PA and to identify amino acids involved in their mechanism of action. The predicted binding pose is fully consistent with the biological data and lays the foundation for the rational development of more effective PA–PB1 inhibitors.     

Bacterial Alarmone,Guanosine 5′‐Diphosphate 3′‐Diphosphate (ppGpp), Predominantly Binds the β′ Subunit of Plastid‐Encoded Plastid RNA Polymerase in Chloroplasts

It's alarming : Bacterial alarmone guanosine 5′‐diphosphate 3′‐diphosphate (ppGpp), which is a key regulatory molecule that controls the stringent response, also exists in chloroplasts of plant cells. Cross‐linking experiments with 6‐thioguanosine 5′‐diphosphate 3′‐diphosphate (6‐thioppGpp) and chloroplast RNA polymerase indicate that ppGpp binds the β′ subunit of plastid‐encoded plastid RNA polymerase that corresponds to the Escherichia coli β′ subunit.

     

自制改性TiO2光催化降解对硝基苯胺的动力学研究

本文采用水热法制备了改性TiO2粉末,用XRD测定了样品的晶型,研究了以自制催化剂对对硝基苯胺的光催化降解效果.通过分析初始浓度、TiO2用量和掺铁量对光催化速率的影响,研究改性TiO2催化对硝基苯胺的动力学行为;结果表明:对硝基苯胺溶液初始浓度为25 mg/L(pH =7)、催化剂用量为0.2 g/L,TiO2掺铁量为0.2％(摩尔分数)、室温下紫外光照(λ =365 nm)反应2.5h,对硝基苯胺的降解率为55.6％,表观反应速率常数,达到最大值0.3152 h-1.光催化反应符合L-H动力学方程,降解过程表现为拟一级反应.