Inhibiting the oligomerization of mycobacterial DNA-directed RNA polymerase(RNAP)using natural compound via in-silico techniques

Mycobacterium tuberculosis(Mtb)is responsible for the spread of tuberculosis(TB).The current study employed virtual screening of 2569 natural compounds against the DNA-directed RNA polymerase(RNAP)of Mtb to identify the possible binders that can inhibit its function.The in-silico methodology included molecular docking to the compounds,further,the stability and flexibility of the best complexes were studied using molecular dynamics simulation,the MM/GBSA binding free energy technique with energy decomposition,PCA,FEL,steered MD simulation,and umbrella sampling.Individual virtual screenings were conducted for the five RNAP subunits(chains A,B,C,D,and E)to identify a compound capable of inhibiting RNAP oligomerization.A promising compound,isoestradiol 3-benzoate,exhibited a low binding score(−7.28kcal/mol to−8.21kcal/mol)and showed binding ability with all subunits of the protein.Thus,the five complexes with isoestradiol 3-benzoate were selected for molecular dynamics simulation analysis.Furthermore,RMSD showed that isoestradiol 3-benzoate bound with chain E showed the lowest RMSD of 0.49nm,while with chains A and B it had the most stable and consistent conformations with RMSD of 1.75nm and 1.2nm,respectively.The H-bond between isoestradiol 3-benzoate and chains C and E showed the highest occupancy(58.27%,45.33%,and 50.80%,42.25%,11.75%).Moreover,the MMPBSA technique showed that isoestradiol 3-benzoate had a strong association with chains B and C(ΔGbind=−126.25±2.03 and−129.27±2.25).Additionally,free energy decomposition,PCA,FEL-steered MD simulation,and umbrella sampling were also performed to validate the association of the ligand with the protein.Isoestradiol 3-benzoate binds strongly to chains B and E;therefore,it should be considered as viable candidate for inhibiting the formation of RNAP protein complex,concluded in this study.

Predictions in Clinical Efficiency of SARS-CoV-2 RNA-Dependent RNA Polymerase (RdRp) Inhibitors by Molecular Docking

This study utilizes the enzyme-substrate complex theory to predict the clinical efficacy of COVID-19 treatments at the biological systems level, using molecular docking stability indicators. Experimental data from the Protein Data Bank and molecular structures generated by AlphaFold 3 were used to create macromolecular complex templates. Six templates were developed, including the holo nsp7-nsp8-nsp12 (RNA-dependent RNA polymerase) complex with dsRNA primers (holo-RdRp-RNA). The study evaluated several ligands—Favipiravir-RTP, Remdesivir, Abacavir, Ribavirin, and Oseltamivir—as potential viral RNA polymerase inhibitors. Notably, the first four of these ligands have been clinically employed in the treatment of COVID-19, allowing for comparative analysis. Molecular docking simulations were performed using AutoDock 4, and statistical differences were assessed through t-tests and Mann-Whitney U tests. A review of the literature on COVID-19 treatment outcomes and inhibitors targeting RNA polymerase enzymes was conducted, and the inhibitors were ranked according to their clinical efficacy: Remdesivir > Favipiravir-RTP > Oseltamivir. Docking results obtained from the second and third templates aligned with clinical observations. Furthermore, Abacavir demonstrated a predicted efficacy comparable to Favipiravir-RTP, while Ribavirin exhibited a predicted efficacy similar to that of Remdesivir. This research, focused on inhibitors of SARS-CoV-2 RNA-dependent RNA polymerase, establishes a framework for screening AI-generated drug templates based on clinical outcomes. Additionally, it develops a drug screening platform based on molecular docking binding energy, enabling the evaluation of novel or repurposed drugs and potentially accelerating the drug development process.

Functional implications of C-terminus of TBX5 with high homology to C-terminal domain of yeast DNA-directed RNA polymerase Ⅱ largest subunit

TBX5, as a member of the T-box-containing transcription factor family, encodes a protein of 518 amino acids and is expressed in the embryonic heart and developing limb tissues.1 The coding region of TBX5 cDNA is 1.5 kb with eight exons including the N-terminal portion, the DNA binding domain and C-terminal region. We reported that the abnormality in transcription level of the TBX5 gene might be the mechanism underlying human simple congenital heart disease in the absence of TBX5 mutations.

血清lncRNA T342620联合AFP对肝癌的诊断价值

目的探究肝癌患者血清长链非编码RNA(lncRNA)T342620的表达水平,及其单独或联合甲胎蛋白(AFP)诊断肝癌的临床应用价值。方法采用病例对照研究,收集2021年4月至2023年5月在中国人民解放军南部战区总医院治疗的69例原发性肝癌患者(肝癌组)、32例乙型肝炎患者(乙肝组)、20例肝硬化患者(肝硬化组)、30例原发性肝癌经导管肝动脉化疗栓塞术(TACE)术后患者(肝癌术后组)和同期进行体检的50例健康者(健康体检组)的血清,提取血清总RNA,采用实时荧光定量PCR技术,检测血清中lncRNA T342620的相对表达量;结合患者临床诊疗资料,分析其表达水平与病理特征和血清学相关指标的相关性;运用受试者工作特征(ROC)曲线分析lncRNA T342620单独及联合AFP对肝癌诊断的特异度和灵敏度。根据ROC曲线下面积(AUC)判断诊断效能,评估其在肝癌诊断中的应用价值。各组间比较采用χ^(2)检验,相关性分析采用Spearman法。结果lncRNA T342620在肝癌组和肝癌术后组血清表达水平较健康体检组、乙肝组、肝硬化组高,差异均具有统计学意义(P<0.001);临床病理和血清学相关指标分析显示:肿瘤越大,血清lncRNAT342620表达水平越高,肝癌组血清lncRNA T342620表达水平与白蛋白(ALB)和白球比(A/G)呈负相关(P<0.05),与α-L-岩藻糖苷酶(AFU)和HBV-DNA呈正相关(P<0.05),肝癌术后组患者血清lncRNA T342620表达水平与总胆汁酸(TBA)呈正相关(P<0.05);ROC曲线分析显示:血清lncRNA T342620用于区别肝癌患者与健康者、乙肝和肝硬化患者时,其灵敏度和特异度分别为55.1%和94.1%,具有较好的诊断价值;和AFP联合检测时,灵敏度和特异度分别为91.3%和91.2%,其灵敏度高于各单项指标诊断的灵敏度,且联合检测诊断效能最高,AUC为0.954,与AFP和lncRNA-T342620单独检测的AUC(0.906、0.758)比较,差异有统计学意义(P<0.05)。结论血清lncRNA T342620有可能成为肝癌辅助诊断的新型血清分子标志物。

polo样激酶1、RNA聚合酶Ⅱ亚基A在人脑胶质瘤中的表达及与预后的关系

目的 探讨polo样激酶1(PLK1)、RNA聚合酶Ⅱ亚基A(POLR2A)在人脑胶质瘤(BG)中的表达及与预后的关系。方法 选取86例BG患者作为观察组,另选取30例非肿瘤脑疾病患者作为参照组,收集观察组患者的BG组织和参照组患者的非肿瘤脑组织。比较两组患者PLK1、POLR2A表达情况和不同临床特征BG患者的BG组织中PLK1、POLR2A的阳性表达情况;比较不同PLK1、POLR2A表达情况BG患者的生存情况;采用Cox回归模型分析BG患者预后的影响因素。结果 观察组患者PLK1、POLR2A阳性表达率分别高于参照组,差异均有统计学意义(P﹤0.05)。低分化BG患者的BG组织中PLK1、POLR2A阳性表达率均高于中高分化患者,差异均有统计学意义(P﹤0.05)。PLK1、POLR2A阳性表达患者的中位生存时间均明显短于PLK1、POLR2A阴性表达患者,差异均有统计学意义(P﹤0.01)。Cox多因素回归分析结果显示,低分化、PLK1阳性表达、POLR2A阳性表达均为BG患者预后的独立危险因素(P﹤0.05)。结论 BG患者PLK1、POLR2A呈高表达,PLK1、POLR2A高表达BG患者中位生存时间缩短,低分化、PLK1阳性表达、POLR2A阳性表达均为BG患者预后的独立危险因素。

重要人感染RNA病毒复制机制和靶向聚合酶药物开发研究进展

近年来,拉沙热、埃博拉、新冠和尼帕等多种病毒性传染病在世界范围内频繁暴发,给人类健康和社会经济发展带来了巨大威胁。广谱抗病毒药物是主动应对新发突发病毒性传染病的重要手段之一,而其研发的关键是发现病毒特有的保守药物靶点。病毒聚合酶负责病毒基因组的复制和转录过程,蛋白序列相对保守,是理想的药物靶点。本文以重要人感染RNA病毒聚合酶的工作机制和靶向病毒聚合酶的药物开发展开综述,为未来新发突发传染性疾病防控提供新思路和新策略。

Systematic identification of endogenous RNA polymeraseⅢpromoters for efficient RNA guidebased genome editing technologies in maize

Single-guide RNA(sg RNA) is one of the two core components of the CRISPR(clustered regularly interspaced short palindromic repeat)/Cas(CRISPR-associated) genome-editing technology. We established an in vitro Traffic Light Reporter(TLR) system, which is designated as the same colors as traffic lights such as green, red and yellow were produced in cells. The TLR can be readily used in maize mesophyll protoplast for a quick test of promoter activity. The TLR assay indicates the variation in transcription activities of the seven Pol III promoters, from 3.4%(U6-1) to over 21.0%(U6-6). The U6-2 promoter, which was constructed to drive sg RNA expression targeting the Zm Wx1 gene, yielded mutation efficiencies ranging from 48.5% to 97.1%. Based on the reported and unpublished data, the in vitro TLR assay results were confirmed to be a readily system and may be extended to other plant species amenable to efficient genome editing via CRISPR/Cas. Our efforts provide an efficient method of identifying native Pol III-recognized promoters for RNA guide-based genome-editing systems in maize.

The VP2 protein of grass carp reovirus(GCRV) expressed in a baculovirus exhibits RNA polymerase activity

The double-shelled grass carp reovirus （GCRV） is capable of endogenous RNA transcription and processing.Genome sequence analysis has revealed that the protein VP2,encoded by gene segment 2 （S2）,is the putative RNA-dependent RNA polymerase （RdRp）.In previous work,we have ex-pressed the functional region of VP2 that is associated with RNA polymerase activity （denoted as rVP2390-900） in E.coil and have prepared a polyclonal antibody against VP2.To characterize the GCRV RNA polymerase,a recombinant full-length VP2 （rVP2） was first constructed and expressed in a baculovirus system,as a fusion protein with an attached His-tag.Immunofluorescence （IF） assays,together with immunoblot （IB） analyses from both expressed cell extracts and purified Histagged rVP2,showed that rVP2 was successfully expressed in Sf9 cells.Further characterization of the replicase activity showed that purified rVP2 and GCRV particles exhibited poly（C）-dependent poly（G） polymerase activity.The RNA enzymatic activity required the divalent cation Mg2＋,and was optimal at 28 ℃.The results provide a foundation for further studies on the RNA polymerases of aquareoviruses during viral transcription and replication.

新型冠状病毒亚基因组RNA检测方法的建立及性能评估

目的建立检测新型冠状病毒(新冠病毒)亚基因组RNA(subgenomic RNA,sgRNA)的方法,并对所建立的方法进行性能评估。方法根据新型冠状病毒sgRNA序列设计引物和探针,建立检测sgRNA的实时荧光逆转录PCR(reverse transcription polymerase chain reaction,RT-PCR)方法,对所建立的方法进行优化,包括引物、探针的浓度及比例、延伸温度、反应体积和模板量。并对所建立的方法进行性能评估,包括最低检测限、灵敏度、特异性和重复性。对临床样本进行新冠病毒sgRNA和基因组RNA(genomic RNA,gRNA)检测,并对检测结果进行分析。结果建立了检测新冠病毒sgRNA的RT-PCR方法。所建方法的最低检测限为100 copies/mL,对常见病原体的检测结果均为阴性。对高、中、低浓度样本sgRNA进行检测,CV均<5%。115例疑似新冠病毒感染者的咽拭子样本sgRNA为阳性(115/330,阳性率为34.85%)。gRNA-N Ct值<30时,sgRNA-N的阳性率为100.00%;gRNA-N的Ct值介于30~32时,sgRNA-N的阳性率为68.75%;gRNA-N的Ct值介于32~35时,sgRNA-N的阳性率为44.44%;gRNA-N的Ct值>35时,sgRNA-N均为阴性。结论建立了新冠病毒sgRNA的RT-PCR检测方法,方法灵敏、特异、重复性好。

Small Amplicons Mutation Library for Vaccine Screening by Error-Prone Polymerase Chain Reaction

Library construction is a common method used to screen target genes in molecular biology.Most library constructions are not suitable for a small DNA library(<100 base pair(bp))and low RNA library output.To maximize the library’s complexity,error-prone polymerase chain reaction(PCR)was used to increase the base mutation rate.After introducing the DNA fragments into the competent cell,the library complexity could reach 109.Library mutation rate increased exponentially with the dilution and amplification of error-prone PCR.The error-prone PCR conditions were optimized including deoxyribonucleotide triphosphate(dNTP)concentration,Mn^(2+)concentration,Mg^(2+)concentration,PCR cycle number,and primer length.Then,a RNA library with high complexity can be obtained by in vitro transcription to meet most molecular biological screening requirements,and can also be used for mRNA vaccine screening.

Hordatines as a Potential Inhibitor of COVID-19 Main Protease and RNA Polymerase: An In-Silico Approach

Total 40 natural compounds were selected to perform the molecular docking studies to screen and identify the potent antiviral agents specifically for Severe Acute Respiratory Syndrome Coronavirus 2 that causes coronavirus disease 2019(COVID-19).The key targets of COVID-19,protease(PDB ID:7BQY)and RNA polymerase(PDB ID:7bV2)were used to dock our target compounds by Molecular Operating Environment(MOE)version 2014.09.We used 3 different conformations of protease target(6M0K,6Y2F and 7BQY)and two different score functions to strengthen the probability of inhibitors discovery.After an extensive screening analysis,20 compounds exhibit good binding affinities to one or both COVID-19 targets.7 out of 20 compounds were predicted to overcome the activity of both targets.The top 7 hits are,flacourticin(3),sagerinic acid(16),hordatine A(23),hordatine B(24),N-feruloyl tyramine dimer(25),bisavenanthramides B-5(29)and vulnibactins(40).According to our results,all these top hits was found to have a better binding scores than remdesivir,the native ligand in RNA polymerase target(PDB ID:7bV2).Hordatines are phenolic compounds present in barley,were found to exhibit the highest binding affinity to both protease and polymerase through forming strong hydrogen bonds with the catalytic residues,as well as significant interactions with other receptor-binding residues.These results probably provided an excellent lead candidate for the development of therapeutic drugs against COVID-19.Eventually,animal experiment and accurate clinical trials are needed to confirm the preventive potentials of these compounds.

流感病毒RNA碱性聚合酶-2抑制剂研究进展

季节流行性感冒是一种易在人际传播的急性病毒感染,是目前对人类健康构成威胁的主要呼吸道传染病之一。迄今为止,流感的预防和治疗主要采用接种疫苗预防和开发抗病毒药物防治并重的应对手段。目前临床应用的抗流感病毒药物包括基质蛋白-2抑制剂(如金刚烷胺)、神经氨酸酶抑制剂(如奥司他韦)、内切酶抑制剂(如巴洛沙韦酯)和聚合酶抑制剂(如法匹拉韦)等,均已产生广泛耐药性。研究开发机制新颖的、与现有药物没有交叉耐药的抗流感病毒药物是解决季节流行性感冒治疗这一尚未完全解决的医学问题,以及应对流感大规模爆发的有效手段。流感病毒RNA碱性聚合酶-2(PB2),由于其在不同流感病毒间的高度保守性而不易产生耐药性,使其成为一个非常有吸引力的药物靶点。目前PB2抑制剂的研究开发已成为抗流感病毒药物的一个热点,特别是围绕明星分子吡莫地韦及其类似物的研究吸引了极大的关注,虽然吡莫地韦因疗效不理想最终止步于Ⅲ期临床试验,但是在其研究过程中所揭示出的PB2蛋白与小分子结合模式及构效关系,为进一步开发该类药物奠定了扎实的基础。本文主要针对PB2抑制剂近10年相关药物化学的研究进展进行综述,并对该药物研究领域的前景提出展望。

Brownian ratchet mechanism of translocation in T7 RNA polymerase facilitated by a post-translocation energy bias arising from the conformational change of the enzyme

T7 RNA polymerase can transcribe DNA to RNA by translocating along the DNA. Structural studies suggest that the pivoting rotation of the O helix in the fingers domain may drive the movement of the O helix C-terminal Tyr639 from pre- to post-translocation positions. In a series of all-atom molecular dynamics simulations, we show that the movement of Tyr639 is not tightly coupled to the rotation of the O helix, and that the two processes are only weakly dependent on each other. We also show that the internal potential of the enzyme itself generates a small difference in free energy （△E） between the post- and pre-translocation positions of Tyr639. The calculated value of △E is consistent with that obtained from single-molecule experimental data. These findings lend support to a model in which the translocation takes place via a Brownian ratchet mechanism, with the small free energy bias △E arising from the conformational change of the enzyme itself.

An intermediate state of T7 RNA polymerase provides another pathway of nucleotide selection

Phage T7 RNA polymerase is a single-subunit transcription enzyme, transcribing template DNA to RNA. Nucleoside triphosphate （NTP） selection and translocation are two critical steps of the transcription elongation. Here, using all-atom molecular dynamics simulations, we found that between pre- and post-translocation states of T7 RNA polymerase an intermediate state exists, where the O helix C-terminal residue tyrosine 639, which plays important roles in translocation, locates between its pre- and post-translocation positions and the side chain of the next template DNA nucleotide has moved into the active site. NTP selection in this intermediate state was studied, revealing that the selection in the intermediate state can be achieved relying on the effect of Watson-Crick interaction between NTP and template DNA nucleotide, effect of stability of the components near the active site such as the nascent DNA-RNA hybrid and role of tyrosine 639. This indicates that another NTP-selection pathway can also exist besides the main pathway where NTP selection begins at the post-translocation state upon the entry of NTE

Analysis of RNA-Dependent RNA Polymerase Sequence of Infectious Flacherie Virus Isolated in China and Its Expression in BmN Cells

Full gene sequence of RNA-dependent RNA polymerase （RdRp） from Bombyx mori infectious flacherie virus isolated in Zhejiang Province, China （Zhejiang01/CHN/2002） was cloned. The sequence was 1 920 nucleotides in length coding 639 amino acid residues. Sequences comparison of RdRp showed Zhejiang01/CHN/2002 was 99.7% nucleotide sequence and 99.1% amino acids sequence homology with Japanese strain. The RdRp sequence was aligned with 8 representative picorna（-like） viruses and 8 highly conserved regions were detected. The result indicated their relevance function. Phylogenetic tree of 14 picorna（-like） viruses which RdRp presumed protein sequences revealed that the viruses from Iflavirus genus formed an independent clade. The RdRp was successfully expressed in BmN cells using Bac-to-Bac expression system.

Probing conformational change of T7 RNA polymerase and DNA complex by solid-state nanopores

Proteins are crucial to most biological processes, such as enzymes, and in various catalytic processes a dynamic motion is required. The dynamics of protein are embodied as a conformational change, which is closely related to the flexibility of protein. Recently, nanopore sensors have become accepted as a low cost and high throughput method to study the features of proteins. In this article, we used a SiN nanopore device to study the flexibility of T7 RNA polymerase（RNAP） and its complex with DNA promoter. By calculating full-width at half-maximum（FWHM） of Gaussian fits to the blockade histograms, we found that T7 RNAP becomes more flexible after binding DNA promoter. Moreover, the distribution of fractional current blockade suggests that flexibility alters due to a breath-like change of the volume.

lncRNA Gm44981抑制心脏衰老的作用和机制研究

目的分析小鼠心脏衰老过程中长链非编码(lnc)RNA Gm44981的表达水平,探索其抑制心脏衰老的机制。方法选取3月龄年轻和24月龄年老小鼠各5只,提取心肌组织RNA后,采用实时定量聚合酶链式反应(qRT-PCR)检测Gm44981的表达水平。另选取4月龄快速老化小鼠10只,分为对照组和Gm44981过表达组(构建Gm44981过表达快速老化小鼠模型),各5只。采用qRT-PCR检测对照组和Gm44981过表达组小鼠Gm44981和衰老相关分泌表型(SASP)表达水平,β-半乳糖苷酶(SA-β-gal)染色检测SA-β-gal活性,蛋白质免疫印迹试验检测p53和Sirt1衰老相关蛋白表达水平。采用qRT-PCR检测miRNA-34a表达水平。结果Gm44981在年老小鼠的心肌组织中表达水平较年轻小鼠低,差异有统计学意义(P<0.05)。与对照组比较,Gm44981过表达组小鼠Gm44981表达上调,白细胞介素(IL)-1α和IL-6等SASP和SA-β-gal表达下调,p53表达下调,Sirt1表达上调,miRNA-34a表达下调,差异均有统计学意义(P<0.05)。结论lncRNA Gm44981在心脏衰老过程中表达下调,其可能通过作用于miRNA-34a/Sirt1抑制心脏衰老。

Snapshots of a Viral RNA Polymerase Switching Gears from Transcription Initiation to Elongation

During transcription initiation,RNA polymerase binds tightly to the promoter DNA defining the start of transcription,transcribes comparatively slowly,and frequently releases short transcripts(3-8 nucleotides)in a process called abortive cycling.Transitioning to elongation,the second phase of transcription,the polymerase dissociates from the promoter while RNA synthesis continues.Elongation is characterized by higher rates of transcription and tight binding to the RNA transcript.The RNA polymerase from enterophage T7 (T7 RNAP) has been used as a model to understand the mechanism of transcription in general,and the transition from initiation to elongation specifically.This single-subunit enzyme undergoes dramatic conformational changes during this transition to support the changing requirements of nucleic acid interactions while continuously maintaining polymerase function.Crystal structures,available of multiple stages of the initiation complex and of the elongation complex,combined with biochemical and biophysical data,offer molecular detail of the transition.Some of the crystal structures contain a variant of T7 RNAP where proline 266 is substituted by leucine.This variant shows less abortive products and altered timing of transition,and is a valuable tool to study these processes.The structural transitions from early to late initiation are well understood and are consistent with solution data.The timing of events and the structural intermediates in the transition from late initiation to elongation are less well understood,but the available data allows one to formulate testable models of the transition to guide further research.

Diversity of the RNA Polymerase in the H7N9 Influenza A Virus

A novel influenza virus of the H7N9 subtype has infected more than 350 people in China since 19 February 2013. Evolutionary analysis indicates that the virus is a reassortant originated from H7, N9 and H9N2 avian influenza viruses, and bears some amino acids associated with mammalian receptor binding, raising concern over the possibility of a new influenza pandemic. Besides HA and NA, the mutation of the polymerase is known to have an important role in virulence, host adaptation and transmissibility in mammalians. In this article, the annotation of the polymerase protein domain associated with molecular function has been highlighted, suggesting the combination of RNA polymerase of H7N9 viruses is still not stable for host adaptation. In addition, the mutation hallmarks in polymerase gene of H7N9 are compared, providing the potential determinants of the evolution in the H7N9 influenza A virus.

A Role for Histone Chaperones in Regulating RNA Polymerase II

Transcription is a highly regulated cellular process in which dysfunction leads to disease. One level of regulation is chromatin structure which protects promoters from transcription factor binding. To circumvent this blockade, histone chaperones aid in displacement of nucleosomes. In particular, the histone chaperone complex HUCA, consisting of Hira, Ubn1, Cabin1, and ASF1a, replaces histone variant H3.1 with H3.3 in front of actively transcribing RNA Polymerase II (RNAPII). The 26S proteasome is a major degrader of proteins within the cell and plays both proteolytic and non- proteolytic roles in transcriptional regulation. One major role is the degradation of irreversibly arrested RNAPII. Several interactions between HUCA, the 26S proteasome, and RNAPII have been characterized individually;we now present observations from our lab and others which directly associate elongating RNAPII with the degradation machinery through observations of involvement with the HUCA complex. Our short report presents these ideas and discusses their importance in transcriptional regulation as well as implications in disease manifestation.