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 Based on the reported and unpublished data, the in vitro TLR assay results were confirmed to be a readily system and may be extended to other plant species amenable to efficient genome editing via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.

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.

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.

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

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.

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.

Promoting the production of challenging proteins via induced expression in CHO cells and modified cell-free lysates harboring T7 RNA polymerase and mutant eIF2α

Chinese hamster ovary(CHO)cells are crucial in biopharmaceutical production due to their scalability and capacity for human-like post-translational modifications.However,toxic proteins and membrane proteins are often difficult-to-express in living cells.Alternatively,cell-free protein synthesis can be employed.This study explores innovative strategies for enhancing the production of challenging proteins through the modification of CHO cells by investigating both,cell-based and cell-free approaches.A major result in our study involves the integration of a mutant eIF2 translation initiation factor and T7 RNA polymerase into CHO cell lysates for cell-free protein synthesis.This resulted in elevated yields,while eliminating the necessity for exogenous additions during cell-free production,thereby substantially enhancing efficiency.Additionally,we explore the potential of the Rosa26 genomic site for the integration of T7 RNA polymerase and cell-based tetracycline-controlled protein expression.These findings provide promising advancements in bioproduction technologies,offering flexibility to switch between cell-free and cell-based protein production as needed.

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.

Crystal structures of RNA-dependent RNA polymerases from Jingmen tick virus and Alongshan virus

Jingmenviruses are a group of flavi-like viruses with segmented genome and have been found in various types of hosts,including humans,cattle,monkeys,bats,rodents,sheep,ticks,mosquitoes and nematodes.Jingmenviruses,including the Jingmen tick virus(JMTV)and Alongshan virus(ALSV),have been associated with febrile illness and flu-like symptoms in humans.Viral polymerase plays critical roles in genome replication and transcription and is an ideal target for antiviral drugs.Here,we determined the crystal structures of RNA-dependent RNA polymerase(RdRp)domains of JMTV and ALSV at 2.6Åand 3.2Åresolutions,respectively.The overall structures of JMTV and ALSV RdRp domains are similar to those from the typical unsegmented viruses in Flaviviridae family,especially the Flavivirus genus.JMTV and ALSV RdRps can be divided into three subdomains and the catalytical Motif A-G are conserved like the typical flaviviruses,whereas the zinc-binding pockets are absent from the JMTV and ALSV RdRps.The 50-ends of jingmenvirus genomes are varied in length and sequence,and a highly conserved 8-nucleotide element located on the tip of stem loop A was identified and shown to be required for binding with RdRp and performing de novo replication activity.These findings provide important structural insights into RdRp of segmented flavivirus and reveal the key region of virus genome responsible for replication initiation,which would promote molecular understanding of segmented flavivirus replication and the structure-based design of antiviral drugs against flaviviruses.

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.

Unusual substructure conformations observed in crystal structures of a dicistrovirus RNA-dependent RNA polymerase suggest contribution of the N-terminal extension in proper folding

The Dicistroviridae is a virus family that includes many insect pathogens.These viruses contain a positive-sense RNA genome that is replicated by the virally encoded RNA-dependent RNA polymerase(RdRP)also named 3D^(pol).Compared with the Picornaviridae RdRPs such as poliovirus(PV)3D^(pol),the Dicistroviridae representative Israeli acute paralysis virus(IAPV)3D^(pol) has an additional N-terminal extension(NE)region that is about 40-residue in length.To date,both the structure and catalytic mechanism of the Dicistroviridae RdRP have remain elusive.Here we reported crystal structures of two truncated forms of IAPV 3D^(pol),namelyΔ85 andΔ40,both missing the NE region,and the 3D^(pol) protein in these structures exhibited three conformational states.The palm and thumb domains of these IAPV 3D^(pol) structures are largely consistent with those of the PV 3D^(pol) structures.However,in all structures,the RdRP fingers domain is partially disordered,while different conformations of RdRP substructures and interactions between them are also present.In particular,a large-scale conformational change occurred in the motif B-middle finger region in one protein chain of theΔ40 structure,while a previously documented alternative conformation of motif A was observed in all IAPV structures.These experimental data on one hand show intrinsic conformational variances of RdRP substructures,and on the other hand suggest possible contribution of the NE region in proper RdRP folding in IAPV.

Equine ANP32 proteins support influenza A virus RNA polymerase activity

Host ANP32 family proteins are crucial for maintaining the activity of influenza RNA polymerase and play an important role in the cross-species transmission of influenza viruses.To date,the molecular properties of equine ANP32(eqANP32)protein are poorly understood,particularly the mechanisms that affect equine influenza virus(EIV)RNA polymerase activity.Here,we found that there are six alternative splicing variants of equine ANP32A(eqANP32A)with different levels of expression.Further studies showed that these six splicing variants of eqANP32A supported the activity of EIV RNA polymerase to varying degrees,with the variant eqANP32A_X2 having the highest expression abundance and exhibiting the highest support of polymerase activity.Sequence analysis demonstrated that the differences in the N-Cap regions of the six splicing variants significantly affected their N-terminal conformation,but did not affect their ability to bind RNA polymerase.We also demonstrated that there is only one transcript of eqANP32B,and that this transcript showed only very low support to the EIV RNA polymerase.This functional defect in eqANP32B is caused by the sequence of the 110–259 amino acids at its Cterminus.Our results indicated that it is the eqANP32A_X2 protein that mainly determines the efficiency of the EIV replication in horses.In conclusion,our study parsed the molecular properties of eqANP32 family proteins and revealed the sequence features of eqANP32A and eqANP32B,suggesting for the first time that the N-Cap region of ANP32A protein also plays an important role in supporting the activity of the influenza virus polymerase.

Structures of EV71 RNA-dependent RNA polymerase in complex with substrate and analogue provide a drug target against the hand-foot-and-mouth disease pandemic in China

Enterovirus 71(EV71),one of the major causative agents for hand-foot-and-mouth disease(HFMD),has caused more than 100 deaths among Chinese children since March 2008.The EV71 genome encodes an RNAdependent RNA polymerase(RdRp),denoted 3D^(pol),which is central for viral genome replication and is a key target for the discovery of specific antiviral therapeutics.Here we report the crystal structures of EV71 RdRp(3D^(pol))and in complex with substrate guanosine-5'-triphosphate and analog 5-bromouridine-5'-triphosphate best to 2.4Åresolution.The structure of EV71 RdRp(3D^(pol))has a wider open thumb domain compared with the most closely related crystal structure of poliovirus RdRp.And the EV71 RdRp(3D^(pol))complex with GTP or Br-UTP bounded shows two distinct movements of the polymerase by substrate or analogue binding.The model of the complex with the template:primer derived by superimposition with foot-and-mouth disease virus(FMDV)3D/RNA complex reveals the likely recognition and binding of template:primer RNA by the polymerase.These results together provide a molecular basis for EV71 RNA replication and reveal a potential target for anti-EV71 drug discovery.

RNA Polymerase V Functions in Arabidopsis Interphase Heterochromatin Organization Independently of the 24-nt siRNA-Directed DNA Methylation Pathway

In Arabidopsis, pericentromeric repeats, retroelements, and silenced rRNA genes are assembled into heterochromatin within nuclear structures known as chromocenters. The mechanisms governing higher-order heterochromatin organization are poorly understood but 24-nt small interfering RNAs （siRNAs） are known to play key roles in heterochromatin formation. Nuclear RNA polymerase IV （Pol IV）, RNA-DEPENDENT RNA POLYMERASE 2 （RDR2）, and DICER-LIKE 3 （DCL3） are required for biogenesis of 24-nt siRNAs that associate with ARGONAUTE 4 （AGO4）. Nuclear RNA polymerase V （Pol V） collaborates with DRD1 （DEFICIENT IN RNA-DEPENDENT DNA METHYLATION 1） to generate transcripts at heterochromatic loci that are hypothesized to bind to siRNA-AGO4 complexes and subsequently recruit the de-novo DNA methylation and/or histone modifying machinery. Here, we report that decondensation of the major pericentromeric repeats and depletion of the heterochromatic mark histone H3 lysine 9 dimethylation at chromocenters occurs specifically in pol V and drdl mutants. Disruption of pericentromeric repeats condensation is coincident with transcriptional reactivation of specific classes of pericentromeric 180-bp repeats. We further demonstrate that Pol V functions independently of Pol IV, RDR2, and DCL3-mediated siRNA production to affect interphase heterochromatin organization, possibly by involving RNAs that recruit structural or chromatin-modifying proteins.

Improved plasmid-based recovery of coxsackievirus A16 infectious clone driven by human RNA polymerase I promoter

Dear Editor,Coxsackievirus A16(CA16)is one of the major viral pathogens associated with hand,foot,and mouth disease.CA16 belongs to the Enterovirus genus of the Picornaviridae family and possesses a single-stranded positivesense RNA genome(Mao et al.,2014).Reverse genetics is an important tool for CA16 research.Previously,a reverse genetics T7 polymerase-based system was de-

SARS-CoV-2 spike protein and RNA dependent RNA polymerase as targets for drug and vaccine development: A review

The present pandemic has posed a crisis to the economy of the world and the health sector.Therefore,the race to expand research to understand some good molecular targets for vaccine and therapeutic development for SARS-CoV-2 is inevitable.The newly discovered coronavirus 2019(COVID-19)is a positive sense,single-stranded RNA,and enveloped virus,assigned to the beta CoV genus.The virus(SARS-CoV-2)is more infectious than the previously detected coronaviruses(MERS and SARS).Findings from many studies have revealed that S protein and RdRp are good targets for drug repositioning,novel therapeutic development(antibodies and small molecule drugs),and vaccine discovery.Therapeutics such as chloroquine,convalescent plasma,monoclonal antibodies,spike binding peptides,and small molecules could alter the ability of S protein to bind to the ACE-2 receptor,and drugs such as remdesivir(targeting SARS-CoV-2 RdRp),favipir,and emetine could prevent SASR-CoV-2 RNA synthesis.The novel vaccines such as mRNA1273(Moderna),3LNP-mRNAs(Pfizer/BioNTech),and ChAdOx1-S(University of Oxford/Astra Zeneca)targeting S protein have proven to be effective in combating the present pandemic.Further exploration of the potential of S protein and RdRp is crucial in fighting the present pandemic.

N-terminal domain of the RNA polymerase of very virulent infectious bursal disease virus contributes to viral replication and virulence

Dear Editor,Infectious bursal disease virus （IBDV） causes an economically significant disease of chickens worldwide （Berg,2000）. The molecular basis for the virulence of very virulent IBDV （vvIBDV） is not fully understood. Previous studies have shown that genome segment A, especically VP2 protein, plays the most important role in the tropism and pathogenicity of serotype 1 IBDV （Brandt et al., 2001）. VP2 is,however, unlikely to be the only factor for the virulence of vvIBDV （Boot et al., 2000）.

Crystal structure of the coxsackievirus A16 RNA-dependent RNA polymerase elongation complex reveals novel features in motif A dynamics

Dear Editor, Coxsackievirus A16 （CV A16） and enterovirus 71 （EV71） are currently the two primary causative agents of hand- foot-and-mouth disease （HFMD） （Solomon et al., 2010; Mao et al., 2014）, threatening health of children world- wide. They both belong to the Enterovirus genus of the Picornaviridae family, and have single-stranded positive- sense RNA genomes of about 7.5 kilobases （kb） in length. As with other positive-strand RNA viruses, the genome rep- lication process ofCV A16 is carried out by a membrane- associated replication complex with the virally encoded RNA-dependent RNA polymerase （RdRP） as the essential catalytic enzyme.