Template Binding Research Articles

Expression and functional study of DNA polymerases from Psychrobacillus sp. BL-248-WT-3 and FJAT-21963.

The properties of DNA polymerases isolated from thermophilic and mesophilic microorganisms, such as the thermophilic Geobacillus stearothermophilus (Bst) and mesophilic Bacillus subtilis phage (Phi29), have been widely researched. However, DNA polymerases in psychrophilic microorganisms remain poorly understood. In this study, we present for the first time the expression and functional characterization of DNA polymerases PWT-WT and FWT-WT from Psychrobacillus sp. BL-248-WT-3 and FJAT-21963. Enzymatic activity assays revealed that FWT-WT possessed strand displacement but lacked exonuclease activity and high ionic strength tolerance, whereas PWT-WT lacked all these properties. Further protein engineering and biochemical analysis identified D423 and S490 as critical mutation sites for improving strand displacement and tolerance to high ionic strength, specifically in the presence of 0-0.3 M potassium chloride (KCl), sodium chloride (NaCl), and potassium acetate (KAc). Three-dimensional structural analysis demonstrated that the size and the electric charge of the single-stranded DNA (ssDNA) encapsulation entrance were pivotal factors in the binding of the ssDNA template.

Overcoming nucleotide bias in the nonenzymatic copying of RNA templates.

The RNA World hypothesis posits that RNA was the molecule of both heredity and function during the emergence of life. This hypothesis implies that RNA templates can be copied, and ultimately replicated, without the catalytic aid of evolved enzymes. A major problem with nonenzymatic template-directed polymerization has been the very poor copying of sequences containing rA and rU. Here, we overcome that problem by using a prebiotically plausible mixture of RNA mononucleotides and random-sequence oligonucleotides, all activated by methyl isocyanide chemistry, that direct the uniform copying of arbitrary-sequence templates, including those harboring rA and rU. We further show that the use of this mixture in copying reactions suppresses copying errors while also generating a more uniform distribution of mismatches than observed for simpler systems. We find that oligonucleotide competition for template binding sites, oligonucleotide ligation and the template binding properties of reactant intermediates work together to reduce product sequence bias and errors. Finally, we show that iterative cycling of templated polymerization and activation chemistry improves the yields of random-sequence products. These results for random-sequence template copying are a significant advance in the pursuit of nonenzymatic RNA replication.

Development of machine learning algorithm for loop-mediated isothermal amplification including influence of temperature

To predict amplification in the Loop-Mediated Isothermal Amplification (LAMP) method, a binary model has been developed. This model assesses whether the combination of partial template binding facilitated by multiple primers aligns with a specific signature indicating amplification. For this model, 1512 settings were adjusted, encompassing variations in the extraction oligomer position from the primer, the oligomer length, the number of allowed mismatched bases, and the stringency of the homology position. The optimal set was determined based on the F1 value at a reaction temperature of 60°C, resulting in a value of 0.58. To implement machine learning by amalgamating multiple models, duplicate models were eliminated through cluster analysis. Machine learning using AutoML was performed on 108 distinct models. Consequently, the F1 values were 0.7119 at a reaction temperature of 60°C and 0.7210 at 68°C. The weak correlation between feature importance and the F1 value of the model alone suggests that machine learning utilizing this model incorporates numerous models to enhance prediction accuracy.

Definition of the binding specificity of the T7 bacteriophage primase by analysis of a protein binding microarray using a thermodynamic model.

Protein binding microarrays (PBM), SELEX, RNAcompeteand chromatin-immunoprecipitation have been intensively used to determine the specificity of nucleic acid binding proteins. While the specificity of proteins with pronounced sequence specificity is straightforward, the determination of the sequence specificity of proteins of modest sequence specificity is more difficult. In this work, an explorative data analysis workflow for nucleic acid binding data was developed that can be used by scientists that want to analyse their binding data. The workflow is based on a regressor realized in scikit-learn, the major machine learning module for the scripting language Python. The regressor is built on a thermodynamic model of nucleic acid binding and describes the sequence specificity with base- and position-specific energies. The regressor was used to determine the binding specificity of the T7 primase. For this, we reanalysed the binding data of the T7 primase obtained with a custom PBM. The binding specificity of the T7 primase agrees with the priming specificity (5'-GTC) and the template (5'-GGGTC) for the preferentially synthesized tetraribonucleotide primer (5'-pppACCC) but is more relaxed. The dominant contribution of two positions in the motif can be explained by the involvement of the initiating and elongating nucleotides for template binding.

Label-free and sensitive fluorescent detection of Escherichia coli O157: H7 in milk based on cascade strand displacement amplification and G-quadruplex-thioflavin T

The development of a simple, rapid, sensitive, and accurate method for detecting Escherichia coli O157:H7 in complex matrices is critical for ensuring food safety. In this study, a fluorescent biosensor employing aptamer (Apt) -functionalized magnetic beads and cascade strand displacement amplification (SDA) was devised for the detection of E. coli O157:H7 in milk samples. E. coli O157:H7 is captured by the aptamer, causing the complementary DNA (cDNA) to not bind to the aptamer and to be released into the supernatant. Following magnetic separation, the cDNA present in the supernatant initiates the opening of the hairpin (HP) structures. This enables primers to attach to the opened HP, triggering the initial SDA process, which generates a substantial quantity of double-stranded DNA (dsDNA) and recycles cDNA to open more HP structures. The dsDNA activates a second SDA, producing single-stranded DNA (ssDNA) for template binding and instigating a third SDA process. Triple SDA leads to the production of numerous G-quadruplex sequences and the formation of a G-quadruplex structure in the presence of K+. The inclusion of ThT into the G-quadruplex significantly enhances fluorescence. Under optimal conditions, this approach can detect E. coli O157:H7 at levels as low as 101 CFU/mL in pure cultures within 2.5 h, with a broad linear detection range of 101–106 CFU/mL. This method exhibits remarkable specificity towards the target bacteria over non-target bacteria. Furthermore, the technique has been effectively utilized for the detection of E. coli O157:H7 in milk samples. Notably, the method involves only two main steps, identification and amplification, significantly reducing contamination risks and minimizing operational steps.

MIP sensor made using multi-current step electro-polymerization method on framed Ag-SPE for glutamic acid enantiomer recognition

A molecularly imprinted, polypyrrole based, sensor with significant enantioselective recognition ability for D- and L- glutamic acids was prepared using a multi-current steps technique on framed silver screened printed electrodes (Ag-SPE). The surfaces of the silver nanoparticles were passivated to obtain a dense, thick MIP film, which was able to inhibit the dissolution of Ag-SPE during the potentiostatic monomer electropolymerization at high positive applied potentials. After template removal, a pretreatment step was carried out at 10 V (vs. Ag/AgCl) to induce template binding and bring about the oxidative dissolution of printed silver particles on the SPE. From the i-t curves, application of the break-down current was found to be needed for only a shorter time for the template-free solution, while no such breakdown was found for the test solution containing the template molecule, implying that the MIP film provided a high resistance that inhibited Ag-SPE dissolution. After pretreatment for 600 s, the optimum selectivities for D- and l-glutamine on their template imprinted films were L/D = (145.9 ± 17.3)/1 and D/L = (126.0 ± 15.4)/1 respectively, based on the current change between 0 and 2 V (vs. Ag/AgCl) with the two enantiomers being present at the same concentration (10 mM). The imprinted glutamic acid films were characterized using scanning electron microscopy with energy dispersive spectroscopy. A recognition mechanism, related to the dissolution of Ag-SPE in the pretreatment step at positive potentials is also proposed.

Structural insights into viral genome replication by the severe fever with thrombocytopenia syndrome virus L protein.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a phenuivirus that has rapidly become endemic in several East Asian countries. The large (L) protein of SFTSV, which includes the RNA-dependent RNA polymerase (RdRp), is responsible for catalysing viral genome replication and transcription. Here, we present 5 cryo-electron microscopy (cryo-EM) structures of the L protein in several states of the genome replication process, from pre-initiation to late-stage elongation, at a resolution of up to 2.6 Å. We identify how the L protein binds the 5' viral RNA in a hook-like conformation and show how the distal 5' and 3' RNA ends form a duplex positioning the 3' RNA terminus in the RdRp active site ready for initiation. We also observe the L protein stalled in the early and late stages of elongation with the RdRp core accommodating a 10-bp product-template duplex. This duplex ultimately splits with the template binding to a designated 3' secondary binding site. The structural data and observations are complemented by in vitro biochemical and cell-based mini-replicon assays. Altogether, our data provide novel key insights into the mechanism of viral genome replication by the SFTSV L protein and will aid drug development against segmented negative-strand RNA viruses.

A remodeled RNA polymerase II complex catalyzing viroid RNA-templated transcription.

Viroids, a fascinating group of plant pathogens, are subviral agents composed of single-stranded circular noncoding RNAs. It is well-known that nuclear-replicating viroids exploit host DNA-dependent RNA polymerase II (Pol II) activity for transcription from circular RNA genome to minus-strand intermediates, a classic example illustrating the intrinsic RNA-dependent RNA polymerase activity of Pol II. The mechanism for Pol II to accept single-stranded RNAs as templates remains poorly understood. Here, we reconstituted a robust in vitro transcription system and demonstrated that Pol II also accepts minus-strand viroid RNA template to generate plus-strand RNAs. Further, we purified the Pol II complex on RNA templates for nano-liquid chromatography-tandem mass spectrometry analysis and identified a remodeled Pol II missing Rpb4, Rpb5, Rpb6, Rpb7, and Rpb9, contrasting to the canonical 12-subunit Pol II or the 10-subunit Pol II core on DNA templates. Interestingly, the absence of Rpb9, which is responsible for Pol II fidelity, explains the higher mutation rate of viroids in comparison to cellular transcripts. This remodeled Pol II is active for transcription with the aid of TFIIIA-7ZF and appears not to require other canonical general transcription factors (such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and TFIIS), suggesting a distinct mechanism/machinery for viroid RNA-templated transcription. Transcription elongation factors, such as FACT complex, PAF1 complex, and SPT6, were also absent in the reconstituted transcription complex. Further analyses of the critical zinc finger domains in TFIIIA-7ZF revealed the first three zinc finger domains pivotal for RNA template binding. Collectively, our data illustrated a distinct organization of Pol II complex on viroid RNA templates, providing new insights into viroid replication, the evolution of transcription machinery, as well as the mechanism of RNA-templated transcription.

In vitro characterisation of the MS2 RNA polymerase complex reveals host factors that modulate emesviral replicase activity

The RNA phage MS2 is one of the most important model organisms in molecular biology and virology. Despite its comprehensive characterisation, the composition of the RNA replication machinery remained obscure. Here, we characterised host proteins required to reconstitute the functional replicase in vitro. By combining a purified replicase sub-complex with elements of an in vitro translation system, we confirmed that the three host factors, EF-Ts, EF-Tu, and ribosomal protein S1, are part of the active replicase holocomplex. Furthermore, we found that the translation initiation factors IF1 and IF3 modulate replicase activity. While IF3 directly competes with the replicase for template binding, IF1 appears to act as an RNA chaperone that facilitates polymerase readthrough. Finally, we demonstrate in vitro formation of RNAs containing minimal motifs required for amplification. Our work sheds light on the MS2 replication machinery and provides a new promising platform for cell-free evolution.

Insights into Binding of Single-Stranded Viral RNA Template to the Replication-Transcription Complex of SARS-CoV-2 for the Priming Reaction from Molecular Dynamics Simulations.

A minimal replication–transcription complex (RTC) of SARS-CoV-2 for synthesis of viral RNAs includes the nsp12 RNA-dependent RNA polymerase and two nsp8 RNA primase subunits for de novo primer synthesis, one nsp8 in complex with its accessory nsp7 subunit and the other without it. The RTC is responsible for faithfully copying the entire (+) sense viral genome from its first 5′-end to the last 3′-end nucleotides through a replication-intermediate (RI) template. The single-stranded (ss) RNA template for the RI is its 33-nucleotide 3′-poly(A) tail adjacent to a well-characterized secondary structure. The ssRNA template for viral transcription is a 5′-UUUAU-3′ next to stem-loop (SL) 1′. We analyze the electrostatic potential distribution of the nsp8 subunit within the RTC around the template strand of the primer/template (P/T) RNA duplex in recently published cryo-EM structures to address the priming reaction using the viral poly(A) template. We carried out molecular dynamics (MD) simulations with a P/T RNA duplex, the viral poly(A) template, or a generic ssRNA template. We find evidence that the viral poly(A) template binds similarly to the template strand of the P/T RNA duplex within the RTC, mainly through electrostatic interactions, providing new insights into the priming reaction by the nsp8 subunit within the RTC, which differs significantly from the existing proposal of the nsp7/nsp8 oligomer formed outside the RTC. High-order oligomerization of nsp8 and nsp7 for SARS-CoV observed outside the RTC of SARS-CoV-2 is not found in the RTC and not likely to be relevant to the priming reaction.

Effects of Hydrostatic Pressure on the Thermodynamics of CspB-Bs Interactions with the ssDNA Template.

Understanding the thermodynamic mechanisms of adaptation of biomacromolecules to high hydrostatic pressure can help shed light on how piezophilic organisms can survive at pressures reaching over 1000 atmospheres. Interaction of proteins with nucleic acids is one of the central processes that allow information flow encoded in the sequence of DNA. Here, we report the results of a study on the interaction of cold shock protein B from Bacillus subtilis (CspB-Bs) with heptadeoxythymine template (pDT7) as a function of temperature and hydrostatic pressure. Experimental data collected at different CspB-Bs:pDT7 ratios were analyzed using a thermodynamic linkage model that accounts for both protein unfolding and CspB-Bs:pDT7 binding. The global fit to the model provided estimates of the stability of CspB-Bs, ΔGProto, the volume change upon CspB-Bs unfolding, ΔVProt, the association constant for CspB-Bs:pDT7 complex, Kao, and the volume changes upon pDT7 single-stranded DNA (ssDNA) template binding, ΔVBind. The protein, CspB-Bs, unfolds with an increase in hydrostatic pressure (ΔVProt < 0). Surprisingly, our study showed that ΔVBind < 0, which means that the binding of CspB-Bs to ssDNA is stabilized by an increase in hydrostatic pressure. Thus, CspB-Bs binding to pDT7 represents a case of linked equilibrium in which folding and binding react differently upon an increase in hydrostatic pressure: protein folding/unfolding equilibrium favors the unfolded state, while protein-ligand binding equilibrium favors the bound state. These opposing effects set a "maximum attainable" pressure tolerance to the protein-ssDNA complex under given conditions.

Stringent Primer Termination by an Archaeo-Eukaryotic DNA Primase.

Priming of single stranded templates is essential for DNA replication. In recent years, significant progress was made in understanding how DNA primase fulfils this fundamental function, particularly with regard to the initiation. Equally intriguing is the unique property of archeao-eukaryotic primases to terminate primer formation at a well-defined unit length. The apparent ability to “count” the number of bases incorporated prior to primer release is not well understood, different mechanisms having been proposed for different species. We report a mechanistic investigation of primer termination by the pRN1 primase from Sulfolobus islandicus. Using an HPLC-based assay we determined structural features of the primer 5′-end that are required for consistent termination. Mutations within the unstructured linker connecting the catalytic domain to the template binding domain allowed us to assess the effect of altered linker length and flexibility on primer termination.

Disruptors, a new class of oligonucleotide reagents, significantly improved PCR performance on templates containing stable intramolecular secondary structures

Intramolecular secondary structures within templates have been shown to lower PCR performance. Whereas many approaches have been developed to mitigate such impairment on PCR, their effects can vary greatly depending on template sequences. Here we present a novel, universally effective approach to improve PCR performance involving specifically designed oligonucleotides called disruptors. A disruptor contained three functional components, an anchor designed to initiate template binding, an effector to disrupt intramolecular secondary structure, and a 3’ blocker to prevent its elongation by DNA polymerase. A functional mechanism for a disruptor to improve PCR efficiency was proposed where anchor first binds to template followed by effector-mediated strand displacement to unwind intramolecular secondary structure. Such a mechanism was consistent with the observation that anchor played a more critical role for disruptor function. As an example of potential disruptor applications, inverted terminal repeat sequences of recombinant adeno-associated virus vectors were successfully amplified in the presence of disruptors despite their well-known reputation as some of the most difficult templates for PCR amplification and Sanger sequencing due to their ultra-stable T-shaped hairpin structures. In stark contrast, both DMSO and betaine, two PCR additives routinely used to facilitate PCR amplification and Sanger sequencing of GC-rich templates, did not demonstrate any improving effect.

Mutations in Animal SARS-CoV-2 Induce Mismatches with the Diagnostic PCR Assays.

Recently, the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was detected in several animal species. After transmission to animals, the virus accumulates mutations in its genome as adaptation to the new animal host progresses. Therefore, we investigated whether these mutations result in mismatches with the diagnostic PCR assays and suggested proper modifications to the oligo sequences accordingly. A comprehensive bioinformatic analysis was conducted using 28 diagnostic PCR assays and 793 publicly available SARS-CoV-2 genomes isolated from animals. Sixteen out of the investigated 28 PCR assays displayed at least one mismatch with their targets at the 0.5% threshold. Mismatches were detected in seven, two, two, and six assays targeting the ORF1ab, spike, envelope, and nucleocapsid genes, respectively. Several of these mismatches, such as the deletions and mismatches at the 3’ end of the primer or probe, are expected to negatively affect the diagnostic PCR assays resulting in false-negative results. The modifications to the oligo sequences should result in stronger template binding by the oligos, better sensitivity of the assays, and higher confidence in the result. It is necessary to monitor the targets of diagnostic PCR assays for any future mutations that may occur as the virus continues to evolve in animals.

Metal-Organic Gel-Modulated Synthesis of Hierarchically Porous Molecularly Imprinted Polymers for Efficient Removal of Sildenafil from Water.

Molecularly imprinted polymers (MIPs) with high specific recognition capability are promising in environmental remediation. However, traditional MIPs usually show poor specific binding affinity toward templates in pure aqueous medium, thus greatly limiting their practical applications in wastewater treatment. Herein, we proposed a facile and versatile method to synthesize a water-compatible hierarchically porous MIP (HP-MIP), in which a metal–organic gel (MOG) was formed by in situ assembly and acted as a removable structural modulator. Remarkably, the integration of the MOG modulator and template imprinting defects significantly improved the specific template binding affinity of HP-MIP in water. The adsorption behavior of HP-MIP fitted well with the heterogeneous Freundlich isotherm, suggesting that HP-MIP possessed greater site heterogeneity to sildenafil than HP-NIP, which confirmed the efficiency of HP-MIP for the removal of sildenafil from water. This approach provides an important pathway to prepare water-compatible porous MIP for efficient removal of highly toxic organic pollutants from wastewater.

Spelling Changes and Fluorescent Tagging With Prime Editing Vectors for Plants.

Prime editing is an adaptation of the CRISPR-Cas system that uses a Cas9(H840A)-reverse transcriptase fusion and a guide RNA amended with template and primer binding site sequences to achieve RNA-templated conversion of the target DNA, allowing specified substitutions, insertions, and deletions. In the first report of prime editing in plants, a variety of edits in rice and wheat were described, including insertions up to 15 bp. Several studies in rice quickly followed, but none reported a larger insertion. Here, we report easy-to-use vectors for prime editing in dicots as well as monocots, their validation in Nicotiana benthamiana, rice, and Arabidopsis, and an insertion of 66 bp that enabled split-GFP fluorescent tagging.

Structural insight into the binding interactions of NTPs and nucleotide analogues to RNA dependent RNA polymerase of SARS-CoV-2

RNA dependent RNA polymerase (RdRP) from positive-stranded RNA viruses has always been a hot target for designing of new drugs. Major class of drugs that are targeted against RdRP are nucleotide analogues. Extensive docking and molecular dynamics study describing the binding of natural nucleotides (NTPs) and its analogues leading to significant structural variation in the RdRP has been presented here. RdRP simulations in its apo, NTP-bound, and analogue-bound form have been performed. Nucleotide analogues included in this study were, favipiravir, galidesivir, lamivudine, ribavirin, remdesivir and sofosbuvir. The conformational flexibility of the RdRP molecule has been explored using principal component (PCA) and Markov state modeling (MSM) analysis. PCA inferred the presence of correlated motions among the conserved motifs of RdRP. Inter-domain distances between the finger and thumb subdomain flanking the nascent RNA template entry site sampled open and closed conformations. The ligand and template binding motifs F and G showed negatively correlated motions. K551, R553, and R555, a part of motif F appear to form strong interactions with the ligand molecules. R836, a primer binding residue was observed to strongly bind to the analogues. MSM analysis helped to extract statistically distinct conformations explored by the RdRP. Ensemble docking of the ligands on the Markov states also suggested the involvement of the above residues in ligand interactions. Markov states obtained clearly demarcated the open/closed conformations of the template entry site. These observations on residues from the conserved motifs involved in binding to the ligands may provide an insight into designing new inhibitors. Communicated by Ramaswamy H. Sarma

Genome-Wide Binding Analyses of HOXB1 Revealed a Novel DNA Binding Motif Associated with Gene Repression.

Knowledge of the diverse DNA binding specificities of transcription factors is important for understanding their specific regulatory functions in animal development and evolution. We have examined the genome-wide binding properties of the mouse HOXB1 protein in embryonic stem cells differentiated into neural fates. Unexpectedly, only a small number of HOXB1 bound regions (7%) correlate with binding of the known HOX cofactors PBX and MEIS. In contrast, 22% of the HOXB1 binding peaks display co-occupancy with the transcriptional repressor REST. Analyses revealed that co-binding of HOXB1 with PBX correlates with active histone marks and high levels of expression, while co-occupancy with REST correlates with repressive histone marks and repression of the target genes. Analysis of HOXB1 bound regions uncovered enrichment of a novel 15 base pair HOXB1 binding motif HB1RE (HOXB1 response element). In vitro template binding assays showed that HOXB1, PBX1, and MEIS can bind to this motif. In vivo, this motif is sufficient for direct expression of a reporter gene and over-expression of HOXB1 selectively represses this activity. Our analyses suggest that HOXB1 has evolved an association with REST in gene regulation and the novel HB1RE motif contributes to HOXB1 function in part through a repressive role in gene expression.

RNA Dependent RNA Polymerases of Severe Acute Respiratory Syndrome-Related Coronaviruses- An Insight into their Active Sites and Mechanism of Action

Aim: To analyze the RNA-dependent RNA polymerases (RdRps) of Severe Acute Respiratory Syndrome (SARS)-related coronaviruses (CoVs) to find out the conserved motifs, metal binding sites and catalytic amino acids and propose a plausible mechanism of action for these enzymes, using SARS-CoV-2 RdRp as a model enzyme.&#x0D; Study Design: Bioinformatics, Biochemical, Site-directed mutagenesis (SDM), X-ray crystallographic and cryo-Electron microscopic (cryo-EM) data were analyzed.&#x0D; Methodology: Bioinformatics, Biochemical, Site-directed mutagenesis, X-ray crystallographic and cryo-EM data of these enzymes from RNA viral pathogens were analyzed. The advanced version of Clustal Omega was used for protein sequence analysis of the RdRps.&#x0D; Results: Multiple sequence alignment (MSA) of RdRps from different SARS-related CoVs show a large number of highly conserved motifs among them. Though the RdRp from the Middle Eastern Respiratory syndrome (MERS)-CoV differed in many conserved regions yet the active site regions are completely conserved. Possible catalytic regions consist of an absolutely conserved amino acid K, as in single subunit (SSU) RNA polymerases and most of the DNA dependent DNA polymerases (DdDps). The invariant ‘gatekeeper/DNA template binding’ YG pair that was reported in all SSU DNA dependent RNA polymerases (DdRps), prokaryotic multi-subunit (MSU) DdRps and DdDps is also highly conserved in the RdRps of SARS-CoVs. The universal metal binding motif –GDD- and an additional motif–SDD- are also found in all SARS-CoV RdRps. In stark contrast, the (–) strand RNA viral pathogens like Ebola, rabies, etc. use –GDN- rather than –GDD- for catalytic metal binding. An invariant YA pair (instead of an YG pair) is found in the primases of the SARS-CoVs. The SARS-CoVs RdRps and primases exhibit very similar active site and catalytic regions with almost same distance conservations between the template binding YG/YA pair and the catalytic K. In SARS-CoV RdRps an invariant R is placed at -5 which is shown to play a role in nucleoside triphosphate (NTP) selection and is in close agreement with SSU DdRps (viral family) and DdDps. In primases no such invariant R/K/H is found very close to the catalytic K in the downstream region, as found in RdRp and Nidovirus RdRp-Associated Nucleotidyltransferase (NiRAN) domains. An invariant YA pair is placed in the NiRAN domain instead of an YG pair, and an invariant H is placed at -5 position. Moreover, the Zn binding motif with the completely conserved Cs and a few DxD/DxxD type metal binding motifs are found in the RdRps and NiRAN domain. However, the primases contained only the DXD type metal binding motifs.&#x0D; Conclusions: The SARS and SARS-related CoV RdRps are very similar as large peptide regions are highly conserved among them. The closer identity between the RdRps of palm civet-CoV and SARS-CoV (CoV-1) suggest their possible link as found between their spike proteins also. The invariant YG and KL pairs may play a role in template binding and catalysis in SARS-CoV RdRps as reported in DdDps. An additional invariant –YAN- motif found in SARS-CoV RdRps may play a crucial role in nucleotide discrimination.

Biochemical Characterization of Respiratory Syncytial Virus RNA-Dependent RNA Polymerase Complex.

RNA-dependent RNA polymerases (RdRPs) from nonsegmented negative strand (NNS) RNA viruses perform both mRNA transcription and genome replication, and these activities are regulated by their interactions with RNA and other accessory proteins within the ribonucleoprotein (RNP) complex. Detailed biochemical characterization of these enzymatic activities and their regulation is essential for understanding the life cycles of many pathogenic RNA viruses and for antiviral drug discovery. We developed biochemical and biophysical kinetic methods to study the RNA synthesis and RNA binding activities of respiratory syncytial virus (RSV) L/P RdRP. We determined that the intact L protein is essential for RdRP activity, and in truncated L protein constructs, RdRP activity is abrogated due to their deficiency in RNA template binding. These results are in agreement with the observation of an RNA template-binding tunnel at the interface of RdRP and capping domains in RSV and vesicular stomatitis virus (VSV) L protein cryo-EM structures. We also describe nonradiometric assays for measuring RNA binding and RNA polymerization activity of RSV RdRP, which are amenable to compound screening and profiling.