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10.1128/mBio.01423-21 http://scihub22266oqcxt.onion/10.1128/mBio.01423-21 34154407!8262916!34154407     free     free     free Deprecated: Implicit conversion from float 213.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 213.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 213.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 213.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 213.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 213.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 247.2 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 247.2 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 247.2 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534       mBio 2021 ; 12 (3): e0142321 Nephropedia Template TP {{tp|p=34154407|t=2021. Allosteric Activation of SARS-CoV-2 RNA-Dependent RNA Polymerase by Remdesivir Triphosphate and Other Phosphorylated Nucleotides |pdf=|usr=}}{{34154407}} gab.com Text Allosteric Activation of SARS-CoV-2 RNA-Dependent RNA Polymerase by Remdesivir Triphosphate and Other Phosphorylated Nucleotides JO: mBio http://www.kidney.de/pget.php?&tw=1&pmid=34154407 #FOAvip The catalytic subunit of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) Nsp12 has a unique nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain that transfers nucleoside monophosphates to the Nsp9 protein and the nascent RNA. The NiRAN and RdRp modules form a dynamic interface distant from their catalytic sites, and both activities are essential for viral replication. We report that codon-optimized (for the pause-free translation in bacterial cells) Nsp12 exists in an inactive state in which NiRAN-RdRp interactions are broken, whereas translation by slow ribosomes and incubation with accessory Nsp7/8 subunits or nucleoside triphosphates (NTPs) partially rescue RdRp activity. Our data show that adenosine and remdesivir triphosphates promote the synthesis of A-less RNAs, as does ppGpp, while amino acid substitutions at the NiRAN-RdRp interface augment activation, suggesting that ligand binding to the NiRAN catalytic site modulates RdRp activity. The existence of allosterically linked nucleotidyl transferase sites that utilize the same substrates has important implications for understanding the mechanism of SARS-CoV-2 replication and the design of its inhibitors. IMPORTANCEIn vitro interrogations of the central replicative complex of SARS-CoV-2, RNA-dependent RNA polymerase (RdRp), by structural, biochemical, and biophysical methods yielded an unprecedented windfall of information that, in turn, instructs drug development and administration, genomic surveillance, and other aspects of the evolving pandemic response. They also illuminated the vast disparity in the methods used to produce RdRp for experimental work and the hidden impact that this has on enzyme activity and research outcomes. In this report, we elucidate the positive and negative effects of codon optimization on the activity and folding of the recombinant RdRp and detail the design of a highly sensitive in vitro assay of RdRp-dependent RNA synthesis. Using this assay, we demonstrate that RdRp is allosterically activated by nontemplating phosphorylated nucleotides, including naturally occurring alarmone ppGpp and synthetic remdesivir triphosphate. Twit Text FOAVip Allosteric Activation of SARS-CoV-2 RNA-Dependent RNA Polymerase by Remdesivir Triphosphate and Other Phosphorylated Nucleotides ????mBio http://www.kidney.de/pget.php?&tw=1&pmid=34154407 #FOAvip Twit Text # Free Paper on # # # # # LINK http://www.kidney.de/pget.php?&tw=1&pmid=34154407 #FOAvip English Wikipedia <ref>{{Cite pmid|34154407}}</ref> Allosteric Activation of SARS-CoV-2 RNA-Dependent RNA Polymerase by Remdesivir Triphosphate and Other Phosphorylated Nucleotides #MMPMID34154407 Wang B; Svetlov V; Wolf YI; Koonin EV; Nudler E; Artsimovitch I mBio 2021[Jun]; 12 (3): e0142321 PMID34154407show ga The catalytic subunit of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) Nsp12 has a unique nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain that transfers nucleoside monophosphates to the Nsp9 protein and the nascent RNA. The NiRAN and RdRp modules form a dynamic interface distant from their catalytic sites, and both activities are essential for viral replication. We report that codon-optimized (for the pause-free translation in bacterial cells) Nsp12 exists in an inactive state in which NiRAN-RdRp interactions are broken, whereas translation by slow ribosomes and incubation with accessory Nsp7/8 subunits or nucleoside triphosphates (NTPs) partially rescue RdRp activity. Our data show that adenosine and remdesivir triphosphates promote the synthesis of A-less RNAs, as does ppGpp, while amino acid substitutions at the NiRAN-RdRp interface augment activation, suggesting that ligand binding to the NiRAN catalytic site modulates RdRp activity. The existence of allosterically linked nucleotidyl transferase sites that utilize the same substrates has important implications for understanding the mechanism of SARS-CoV-2 replication and the design of its inhibitors. IMPORTANCEIn vitro interrogations of the central replicative complex of SARS-CoV-2, RNA-dependent RNA polymerase (RdRp), by structural, biochemical, and biophysical methods yielded an unprecedented windfall of information that, in turn, instructs drug development and administration, genomic surveillance, and other aspects of the evolving pandemic response. They also illuminated the vast disparity in the methods used to produce RdRp for experimental work and the hidden impact that this has on enzyme activity and research outcomes. In this report, we elucidate the positive and negative effects of codon optimization on the activity and folding of the recombinant RdRp and detail the design of a highly sensitive in vitro assay of RdRp-dependent RNA synthesis. Using this assay, we demonstrate that RdRp is allosterically activated by nontemplating phosphorylated nucleotides, including naturally occurring alarmone ppGpp and synthetic remdesivir triphosphate. |Adenosine Triphosphate/*analogs & derivatives/pharmacology[MESH] |Antiviral Agents/*pharmacology[MESH] |COVID-19 Drug Treatment[MESH] |Catalytic Domain/physiology[MESH] |Coronavirus RNA-Dependent RNA Polymerase/genetics/*metabolism[MESH] |Guanosine Tetraphosphate/*pharmacology[MESH] |Humans[MESH] |Ribosomes/metabolism[MESH]Allosteric Activation of SARS-CoV-2 RNA-Dependent RNA Polymerase by Re(epmc).pdf DeepDyve Pubget Overpricing