3DFlex: determining structure and motion of flexible proteins from cryo-EM.

Modeling flexible macromolecules is one of the foremost challenges in single-particle cryogenic-electron microscopy (cryo-EM), with the potential to illuminate fundamental questions in structural biology. We introduce Three-Dimensional Flexible Refinement (3DFlex), a motion-based neural network model for continuous molecular heterogeneity for cryo-EM data. 3DFlex exploits knowledge that conformational variability of a protein is often the result of physical processes that transport density over space and tend to preserve local geometry. From two-dimensional image data, 3DFlex enables the determination of high-resolution 3D density, and provides an explicit model of a flexible protein's motion over its conformational landscape. Experimentally, for large molecular machines (tri-snRNP spliceosome complex, translocating ribosome) and small flexible proteins (TRPV1 ion channel, αVß8 integrin, SARS-CoV-2 spike), 3DFlex learns nonrigid molecular motions while resolving details of moving secondary structure elements. 3DFlex can improve 3D density resolution beyond the limits of existing methods because particle images contribute coherent signal over the conformational landscape.

Selective translational control of cellular and viral mRNAs by RPS3 mRNA binding.

RPS3, a universal core component of the 40S ribosomal subunit, interacts with mRNA at the entry channel. Whether RPS3 mRNA-binding contributes to specific mRNA translation and ribosome specialization in mammalian cells is unknown. Here we mutated RPS3 mRNA-contacting residues R116, R146 and K148 and report their impact on cellular and viral translation. R116D weakened cap-proximal initiation and promoted leaky scanning, while R146D had the opposite effect. Additionally, R146D and K148D displayed contrasting effects on start-codon fidelity. Translatome analysis uncovered common differentially translated genes of which the downregulated set bears long 5'UTR and weak AUG context, suggesting a stabilizing role during scanning and AUG selection. We identified an RPS3-dependent regulatory sequence (RPS3RS) in the sub-genomic 5'UTR of SARS-CoV-2 consisting of a CUG initiation codon and a downstream element that is also the viral transcription regulatory sequence (TRS). Furthermore, RPS3 mRNA-binding residues are essential for SARS-CoV-2 NSP1-mediated inhibition of host translation and for its ribosomal binding. Intriguingly, NSP1-induced mRNA degradation was also reduced in R116D cells, indicating that mRNA decay occurs in the ribosome context. Thus, RPS3 mRNA-binding residues have multiple translation regulatory functions and are exploited by SARS-CoV-2 in various ways to influence host and viral mRNA translation and stability.

Genome-Scale CRISPR Screening Reveals Host Factors Required for Ribosome Formation and Viral Replication.

Viruses are known to co-opt host machinery for translation initiation, but less is known about which host factors are required for the formation of ribosomes used to synthesize viral proteins. Using a loss-of-function CRISPR screen, we show that synthesis of a flavivirus-encoded fluorescent reporter depends on multiple host factors, including several 60S ribosome biogenesis proteins. Viral phenotyping revealed that two of these factors, SBDS, a known ribosome biogenesis factor, and the relatively uncharacterized protein SPATA5, were broadly required for replication of flaviviruses, coronaviruses, alphaviruses, paramyxoviruses, an enterovirus, and a poxvirus. Mechanistic studies revealed that loss of SPATA5 caused defects in rRNA processing and ribosome assembly, suggesting that this human protein may be a functional ortholog of yeast Drg1. These studies implicate specific ribosome biogenesis proteins as viral host dependency factors that are required for synthesis of virally encoded protein and accordingly, optimal viral replication. IMPORTANCE Viruses are well known for their ability to co-opt host ribosomes to synthesize viral proteins. The specific factors involved in translation of viral RNAs are not fully described. In this study, we implemented a unique genome-scale CRISPR screen to identify previously uncharacterized host factors that are important for the synthesis of virally encoded protein. We found that multiple genes involved in 60S ribosome biogenesis were required for viral RNA translation. Loss of these factors severely impaired viral replication. Mechanistic studies on the AAA ATPase SPATA5 indicate that this host factor is required for a late step in ribosome formation. These findings reveal insight into the identity and function of specific ribosome biogenesis proteins that are critical for viral infections.

Ribosome biogenesis in disease: new players and therapeutic targets.

The ribosome is a multi-unit complex that translates mRNA into protein. Ribosome biogenesis is the process that generates ribosomes and plays an essential role in cell proliferation, differentiation, apoptosis, development, and transformation. The mTORC1, Myc, and noncoding RNA signaling pathways are the primary mediators that work jointly with RNA polymerases and ribosome proteins to control ribosome biogenesis and protein synthesis. Activation of mTORC1 is required for normal fetal growth and development and tissue regeneration after birth. Myc is implicated in cancer development by enhancing RNA Pol II activity, leading to uncontrolled cancer cell growth. The deregulation of noncoding RNAs such as microRNAs, long noncoding RNAs, and circular RNAs is involved in developing blood, neurodegenerative diseases, and atherosclerosis. We review the similarities and differences between eukaryotic and bacterial ribosomes and the molecular mechanism of ribosome-targeting antibiotics and bacterial resistance. We also review the most recent findings of ribosome dysfunction in COVID-19 and other conditions and discuss the consequences of ribosome frameshifting, ribosome-stalling, and ribosome-collision. We summarize the role of ribosome biogenesis in the development of various diseases. Furthermore, we review the current clinical trials, prospective vaccines for COVID-19, and therapies targeting ribosome biogenesis in cancer, cardiovascular disease, aging, and neurodegenerative disease.

Translational Control of COVID-19 and Its Therapeutic Implication.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, which has broken out worldwide for more than two years. However, due to limited treatment, new cases of infection are still rising. Therefore, there is an urgent need to understand the basic molecular biology of SARS-CoV-2 to control this virus. SARS-CoV-2 replication and spread depend on the recruitment of host ribosomes to translate viral messenger RNA (mRNA). To ensure the translation of their own mRNAs, the SARS-CoV-2 has developed multiple strategies to globally inhibit the translation of host mRNAs and block the cellular innate immune response. This review provides a comprehensive picture of recent advancements in our understanding of the molecular basis and complexity of SARS-CoV-2 protein translation. Specifically, we summarize how this viral infection inhibits host mRNA translation to better utilize translation elements for translation of its own mRNA. Finally, we discuss the potential of translational components as targets for therapeutic interventions.

Prevention of ribosome collision-induced neuromuscular degeneration by SARS CoV-2-encoded Nsp1.

An overarching goal of aging and age-related neurodegenerative disease research is to discover effective therapeutic strategies applicable to a broad spectrum of neurodegenerative diseases. Little is known about the extent to which targetable pathogenic mechanisms are shared among these seemingly diverse diseases. Translational control is critical for maintaining proteostasis during aging. Gaining control of the translation machinery is also crucial in the battle between viruses and their hosts. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing COVID-19 pandemic. Here, we show that overexpression of SARS-CoV-2-encoded nonstructural protein 1 (Nsp1) robustly rescued neuromuscular degeneration and behavioral phenotypes in Drosophila models of Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. These diseases share a common mechanism: the accumulation of aberrant protein species due to the stalling and collision of translating ribosomes, leading to proteostasis failure. Our genetic and biochemical analyses revealed that Nsp1 acted in a multipronged manner to resolve collided ribosomes, abort stalled translation, and remove faulty translation products causative of disease in these models, at least in part through the ribosome recycling factor ABCE1, ribosome-associated quality-control factors, autophagy, and AKT signaling. Nsp1 exhibited exquisite specificity in its action, as it did not modify other neurodegenerative conditions not known to be associated with ribosome stalling. These findings uncover a previously unrecognized mechanism of Nsp1 in manipulating host translation, which can be leveraged for combating age-related neurodegenerative diseases that are affecting millions of people worldwide and currently without effective treatment.

Abracadabra, One Becomes Two: The Importance of Context in Viral -1 Programmed Ribosomal Frameshifting.

The constrained nature of viral genomes has allowed a translational sleight of hand known as -1 Programmed Ribosomal Frameshifting (-1 PRF) to flourish. Numerous studies have sought to tease apart the mechanisms and implications of -1PRF utilizing a few techniques. The dual-luciferase assay and ribosomal profiling have driven the PRF field to make great advances; however, the use of these assays means that the full impact of the genomic and cellular context on -1 PRF is often lost. Here, we discuss how the Minimal Frameshifting Element (MFE) and its constraints can hide contextual effects on -1 PRF. We review how sequence elements proximal to the traditionally defined MFE, such as the coronavirus attenuator sequence, can affect the observed rates of -1 PRF. Further, the MFE-based approach fully obscured -1 PRF in Barley yellow dwarf virus and would render the exploration of -1 PRF difficult in Porcine reproductive and respiratory syndrome virus, Encephalomyocarditis virus, Theiler's murine encephalomyelitis virus, and Sindbis virus. Finally, we examine how the cellular context of tRNA abundance, miRNAs, and immune response elements can affect -1 PRF. The use of MFE was instrumental in establishing the basic foundations of PRF; however, it has become clear that the contextual impact on -1 PRF is no longer the exception so much as it is the rule and argues for new approaches to study -1PRF that embrace context. We therefore urge our field to expand the strategies and methods used to explore -1 PRF.

The Translational Landscape of SARS-CoV-2-infected Cells Reveals Suppression of Innate Immune Genes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes a number of strategies to modulate viral and host mRNA translation. Here, we used ribosome profiling in SARS-CoV-2-infected model cell lines and primary airway cells grown at an air-liquid interface to gain a deeper understanding of the translationally regulated events in response to virus replication. We found that SARS-CoV-2 mRNAs dominate the cellular mRNA pool but are not more efficiently translated than cellular mRNAs. SARS-CoV-2 utilized a highly efficient ribosomal frameshifting strategy despite notable accumulation of ribosomes within the slippery sequence on the frameshifting element. In a highly permissive cell line model, although SARS-CoV-2 infection induced the transcriptional upregulation of numerous chemokine, cytokine, and interferon-stimulated genes, many of these mRNAs were not translated efficiently. The impact of SARS-CoV-2 on host mRNA translation was more subtle in primary cells, with marked transcriptional and translational upregulation of inflammatory and innate immune responses and downregulation of processes involved in ciliated cell function. Together, these data reveal the key role of mRNA translation in SARS-CoV-2 replication and highlight unique mechanisms for therapeutic development. IMPORTANCE SARS-CoV-2 utilizes a number of strategies to modulate host responses to ensure efficient propagation. Here, we used ribosome profiling in SARS-CoV-2-infected cells to gain a deeper understanding of the translationally regulated events in infected cells. We found that although viral mRNAs are abundantly expressed, they are not more efficiently translated than cellular mRNAs. SARS-CoV-2 utilized a highly efficient ribosomal frameshifting strategy and alternative translation initiation sites that help increase the coding potential of its RNAs. In permissive cells, SARS-CoV-2 infection induced the translational repression of numerous innate immune mediators. Though the impact of SARS-CoV-2 on host mRNA translation was more subtle in primary airway cell cultures, we noted marked transcriptional and translational upregulation of inflammatory and innate immune responses and downregulation of processes involved in ciliated cell function. Together, these data provide new insight into how SARS-CoV-2 modulates innate host responses and highlight unique mechanisms for therapeutic intervention.

Clinically observed deletions in SARS-CoV-2 Nsp1 affect its stability and ability to inhibit translation.

Nonstructural protein 1 (Nsp1) of SARS-CoV-2 inhibits host cell translation through an interaction between its C-terminal domain and the 40S ribosome. The N-terminal domain (NTD) of Nsp1 is a target of recurring deletions, some of which are associated with altered COVID-19 disease progression. Here, we characterize the efficiency of translational inhibition by clinically observed Nsp1 deletion variants. We show that a frequent deletion of residues 79-89 severely reduces the ability of Nsp1 to inhibit translation while not abrogating Nsp1 binding to the 40S. Notably, while the SARS-CoV-2 5' untranslated region enhances translation of mRNA, it does not protect from Nsp1-mediated inhibition. Finally, thermal stability measurements and structure predictions reveal a correlation between stability of the NTD and the efficiency of translation inhibition.

The key features of SARS-CoV-2 leader and NSP1 required for viral escape of NSP1-mediated repression.

SARS-CoV-2, responsible for the ongoing global pandemic, must overcome a conundrum faced by all viruses. To achieve its own replication and spread, it simultaneously depends on and subverts cellular mechanisms. At the early stage of infection, SARS-CoV-2 expresses the viral nonstructural protein 1 (NSP1), which inhibits host translation by blocking the mRNA entry tunnel on the ribosome; this interferes with the binding of cellular mRNAs to the ribosome. Viral mRNAs, on the other hand, overcome this blockade. We show that NSP1 enhances expression of mRNAs containing the SARS-CoV-2 leader. The first stem-loop (SL1) in the viral leader is both necessary and sufficient for this enhancement mechanism. Our analysis pinpoints specific residues within SL1 (three cytosine residues at the positions 15, 19, and 20) and another within NSP1 (R124), which are required for viral evasion, and thus might present promising drug targets. We target SL1 with the antisense oligo (ASO) to efficiently and specifically down-regulate SARS-CoV-2 mRNA. Additionally, we carried out analysis of a functional interactome of NSP1 using BioID and identified components of antiviral defense pathways. Our analysis therefore suggests a mechanism by which NSP1 inhibits the expression of host genes while enhancing that of viral RNA. This analysis helps reconcile conflicting reports in the literature regarding the mechanisms by which the virus avoids NSP1 silencing.

Chemical modifications to mRNA nucleobases impact translation elongation and termination.

Messenger RNAs (mRNAs) serve as blueprints for protein synthesis by the molecular machine the ribosome. The ribosome relies on hydrogen bonding interactions between adaptor aminoacyl-transfer RNA molecules and mRNAs to ensure the rapid and faithful translation of the genetic code into protein. There is a growing body of evidence suggesting that chemical modifications to mRNA nucleosides impact the speed and accuracy of protein synthesis by the ribosome. Modulations in translation rates have downstream effects beyond protein production, influencing protein folding and mRNA stability. Given the prevalence of such modifications in mRNA coding regions, it is imperative to understand the consequences of individual modifications on translation. In this review we present the current state of our knowledge regarding how individual mRNA modifications influence ribosome function. Our comprehensive comparison of the impacts of 16 different mRNA modifications on translation reveals that most modifications can alter the elongation step in the protein synthesis pathway. Additionally, we discuss the context dependence of these effects, highlighting the necessity of further study to uncover the rules that govern how any given chemical modification in an mRNA codon is read by the ribosome.

Reconstitution of a mini-gene cluster combined with ribosome engineering led to effective enhancement of salinomycin production in Streptomyces albus.

Salinomycin, an FDA-approved polyketide drug, was recently identified as a promising anti-tumour and anti-viral lead compound. It is produced by Streptomyces albus, and the biosynthetic gene cluster (sal) spans over 100 kb. The genetic manipulation of large polyketide gene clusters is challenging, and approaches delivering reliable efficiency and accuracy are desired. Herein, a delicate strategy to enhance salinomycin production was devised and evaluated. We reconstructed a minimized sal gene cluster (mini-cluster) on pSET152 including key genes responsible for tailoring modification, antibiotic resistance, positive regulation and precursor supply. These genes were overexpressed under the control of constitutive promoter PkasO* or Pneo . The pks operon was not included in the mini-cluster, but it was upregulated by SalJ activation. After the plasmid pSET152::mini-cluster was introduced into the wild-type strain and a chassis host strain obtained by ribosome engineering, salinomycin production was increased to 2.3-fold and 5.1-fold compared with that of the wild-type strain respectively. Intriguingly, mini-cluster introduction resulted in much higher production than overexpression of the whole sal gene cluster. The findings demonstrated that reconstitution of sal mini-cluster combined with ribosome engineering is an efficient novel approach and may be extended to other large polyketide biosynthesis.

Nsp1 of SARS-CoV-2 stimulates host translation termination.

Nsp1 of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of the Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1, eRF1 and ABCE1. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex, we hypothesized that Nsp1 may be involved in translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we show that Nsp1 stimulates peptide release and formation of termination complexes. Detailed analysis of Nsp1 activity during translation termination stages reveals that Nsp1 facilitates stop codon recognition. We demonstrate that Nsp1 stimulation targets eRF1 and does not affect eRF3. Moreover, Nsp1 increases amount of the termination complexes at all three stop codons. The activity of Nsp1 in translation termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that the biological meaning of Nsp1 activity in translation termination is binding with the 80S ribosomes translating host mRNAs and remove them from the pool of the active ribosomes.

SMOG 2 and OpenSMOG: Extending the limits of structure-based models.

Applying simulations with structure-based Go¯-like models has proven to be an effective strategy for investigating the factors that control biomolecular dynamics. The common element of these models is that some (or all) of the intra/inter-molecular interactions are explicitly defined to stabilize an experimentally determined structure. To facilitate the development and application of this broad class of models, we previously released the SMOG 2 software package. This suite allows one to easily customize and distribute structure-based (i.e., SMOG) models for any type of polymer-ligand system. The force fields generated by SMOG 2 may then be used to perform simulations in highly optimized MD packages, such as Gromacs, NAMD, LAMMPS, and OpenMM. Here, we describe extensions to the software and demonstrate the capabilities of the most recent version (SMOG v2.4.2). Changes include new tools that aid user-defined customization of force fields, as well as an interface with the OpenMM simulation libraries (OpenSMOG v1.1.0). The OpenSMOG module allows for arbitrary user-defined contact potentials and non-bonded potentials to be employed in SMOG models, without source-code modifications. To illustrate the utility of these advances, we present applications to systems with millions of atoms, long polymers and explicit ions, as well as models that include non-structure-based (e.g., AMBER-based) energetic terms. Examples include large-scale rearrangements of the SARS-CoV-2 Spike protein, the HIV-1 capsid with explicit ions, and crystallographic lattices of ribosomes and proteins. In summary, SMOG 2 and OpenSMOG provide robust support for researchers who seek to develop and apply structure-based models to large and/or intricate biomolecular systems.

Proteomics reveal cap-dependent translation inhibitors remodel the translation machinery and translatome.

Tactical disruption of protein synthesis is an attractive therapeutic strategy, with the first-in-class eIF4A-targeting compound zotatifin in clinical evaluation for cancer and COVID-19. The full cellular impact and mechanisms of these potent molecules are undefined at a proteomic level. Here, we report mass spectrometry analysis of translational reprogramming by rocaglates, cap-dependent initiation disruptors that include zotatifin. We find effects to be far more complex than simple "translational inhibition" as currently defined. Translatome analysis by TMT-pSILAC (tandem mass tag-pulse stable isotope labeling with amino acids in cell culture mass spectrometry) reveals myriad upregulated proteins that drive hitherto unrecognized cytotoxic mechanisms, including GEF-H1-mediated anti-survival RHOA/JNK activation. Surprisingly, these responses are not replicated by eIF4A silencing, indicating a broader translational adaptation than currently understood. Translation machinery analysis by MATRIX (mass spectrometry analysis of active translation factors using ribosome density fractionation and isotopic labeling experiments) identifies rocaglate-specific dependence on specific translation factors including eEF1ε1 that drive translatome remodeling. Our proteome-level interrogation reveals that the complete cellular response to these historical "translation inhibitors" is mediated by comprehensive translational landscape remodeling.

The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in suppression of cellular gene expression and preservation of viral gene expression.

Nonstructural protein 1 (nsp1) is a coronavirus (CoV) virulence factor that restricts cellular gene expression by inhibiting translation through blocking the mRNA entry channel of the 40S ribosomal subunit and by promoting mRNA degradation. We perform a detailed structure-guided mutational analysis of severe acute respiratory syndrome (SARS)-CoV-2 nsp1, revealing insights into how it coordinates these activities against host but not viral mRNA. We find that residues in the N-terminal and central regions of nsp1 not involved in docking into the 40S mRNA entry channel nonetheless stabilize its association with the ribosome and mRNA, both enhancing its restriction of host gene expression and enabling mRNA containing the SARS-CoV-2 leader sequence to escape translational repression. These data support a model in which viral mRNA binding functionally alters the association of nsp1 with the ribosome, which has implications for drug targeting and understanding how engineered or emerging mutations in SARS-CoV-2 nsp1 could attenuate the virus.

I(nsp1)ecting SARS-CoV-2-ribosome interactions.

While SARS-CoV-2 is causing modern human history's most serious health crisis and upending our way of life, clinical and basic research on the virus is advancing rapidly, leading to fascinating discoveries. Two studies have revealed how the viral virulence factor, nonstructural protein 1 (Nsp1), binds human ribosomes to inhibit host cell translation. Here, we examine the main conclusions on the molecular activity of Nsp1 and its role in suppressing innate immune responses. We discuss different scenarios potentially explaining how the viral RNA can bypass its own translation blockage and speculate on the suitability of Nsp1 as a therapeutic target.

The SARS-CoV-2 Programmed -1 Ribosomal Frameshifting Element Crystal Structure Solved to 2.09 Å Using Chaperone-Assisted RNA Crystallography.

The programmed -1 ribosomal frameshifting element (PFSE) of SARS-CoV-2 is a well conserved structured RNA found in all coronaviruses' genomes. By adopting a pseudoknot structure in the presence of the ribosome, the PFSE promotes a ribosomal frameshifting event near the stop codon of the first open reading frame Orf1a during translation of the polyprotein pp1a. Frameshifting results in continuation of pp1a via a new open reading frame, Orf1b, that produces the longer pp1ab polyprotein. Polyproteins pp1a and pp1ab produce nonstructural proteins NSPs 1-10 and NSPs 1-16, respectively, which contribute vital functions during the viral life cycle and must be present in the proper stoichiometry. Both drugs and sequence alterations that affect the stability of the -1 programmed ribosomal frameshifting element disrupt the stoichiometry of the NSPs produced, which compromise viral replication. For this reason, the -1 programmed frameshifting element is considered a promising drug target. Using chaperone assisted RNA crystallography, we successfully crystallized and solved the three-dimensional structure of the PFSE. We observe a three-stem H-type pseudoknot structure with the three stems stacked in a vertical orientation stabilized by two triple base pairs at the stem 1/stem 2 and stem 1/stem 3 junctions. This structure provides a new conformation of PFSE distinct from the bent conformations inferred from midresolution cryo-EM models and provides a high-resolution framework for mechanistic investigations and structure-based drug design.

Structural dynamics of single SARS-CoV-2 pseudoknot molecules reveal topologically distinct conformers.

The RNA pseudoknot that stimulates programmed ribosomal frameshifting in SARS-CoV-2 is a possible drug target. To understand how it responds to mechanical tension applied by ribosomes, thought to play a key role during frameshifting, we probe its structural dynamics using optical tweezers. We find that it forms multiple structures: two pseudoknotted conformers with different stability and barriers, and alternative stem-loop structures. The pseudoknotted conformers have distinct topologies, one threading the 5' end through a 3-helix junction to create a knot-like fold, the other with unthreaded 5' end, consistent with structures observed via cryo-EM and simulations. Refolding of the pseudoknotted conformers starts with stem 1, followed by stem 3 and lastly stem 2; Mg2+ ions are not required, but increase pseudoknot mechanical rigidity and favor formation of the knot-like conformer. These results resolve the SARS-CoV-2 frameshift signal folding mechanism and highlight its conformational heterogeneity, with important implications for structure-based drug-discovery efforts.

Allosteric Activation of SARS-CoV-2 RNA-Dependent RNA Polymerase by Remdesivir Triphosphate and Other Phosphorylated Nucleotides.

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.