Emerging Trends and Research in Biological Science Vol. 1

Palanivelu, Peramachi; Prasad, Aarathi; Osna, Natalia A.; Ganesan, M.; Poluektova, Larisa Y.; Kharbanda, Kusum K.; Ergüden, Deniz; Ayas, Deniz; Ergüden, Sibel Alagöz; Akbora, Hasan Deniz; Mathieu, N.; Blandine, Mezajoug Kenfack Laurette; Gouado, Inocent; Tchiégang, C.; Jeanne, Ngogang; Byadgi, Shameembanu A.; Kulloli, Sadhana D.; Venugopal, C. K.; Bala, Rajni

doi:10.9734/bpi/etrbs/v1

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“…5). The catalytic region and catalytic amino acid of the CoV-2 RdRps were arrived at from the MSA and cryo-EM data [17], and also from the data already available on other RdRps, DdRps and DNA polymerases [22][23][24][25][26]. The prokaryotic and eukaryotic multi-subunit (MSU) DdRp catalytic subunits invariably use the catalytic amino acid R followed by an invariant Y/FG (except human RNA polymerase where it uses a KG) whereas the single subunit (SSU) RNA polymerases and DNA polymerases mostly use the catalytic amino acid K followed by an invariant YG pair (Table 1).…”

Section: Analyses Of Active Site Regions Of Rdrps Of Sars-covsmentioning

confidence: 99%

“…MSA analysis of the SARS and SARS-related CoVs uses an invariant K which is followed by the invariant YG pair where the invariant K could be possibly the catalytic amino acid as in DdDps (Table 1). The YG which is commonly found in all these nucleic acid polymerases is implicated in template binding [22][23][24][25]. Wang et al [2] investigated the molecular basis of SARS-CoV-2 RNA replication by analyzing the cryo-EM structures of the stalled pre-and post-translocated polymerase complexes.…”

Section: Analyses Of Active Site Regions Of Rdrps Of Sars-covsmentioning

confidence: 99%

“…However, the MSU DdRps of both prokaryotic (eubacterial and chloroplasts) and eukaryotic organisms use a different metal binding sites like -E/DxD-, -NxEDS-(for initiation reaction) and -NADFDGD-(for elongation reaction) (Table 2) [24][25][26]40]. By electron paramagnetic resonance spectroscopy, flow-dialysis and transcription analysis, the D 537 and D 812 in bacteriophage T7 RNA polymerase were found to be as metal ion-binding sites and are found to be essential for the catalytic mechanism [41].…”

Section: Analyses Of Metal Binding Sites In Rdrps Of + and -Strand Rna Viral Pathogensmentioning

confidence: 99%

“…It is interesting to note that all the observed structural features of DNA and RNA polymerases suggest not only a strong conservation of structure-function relationships among them, but also a very similar active site amino acids and catalytic mechanism [22][23][24][25][26]. A minimal number of steps involved in the catalytic cycle of RdRps are: NTP selection, Watson-Crick base pairing with the complementary nucleotide to the template, catalysis, pyrophosphate release and translocation.…”

Section: Mechanism Of Action Of the Rdrp Of Sars-cov-2mentioning

confidence: 99%

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

Palanivelu

2020

IJBcRR

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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. Study Design: Bioinformatics, Biochemical, Site-directed mutagenesis (SDM), X-ray crystallographic and cryo-Electron microscopic (cryo-EM) data were analyzed. 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. 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. 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.

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Section: Analyses Of Active Site Regions Of Rdrps Of Sars-covsmentioning

confidence: 99%