The SARS-CoV-2 RNA-dependent RNA polymerase coordinates viral RNA synthesis as part of an assembly known as the replication-transcription complex (RTC) 1 . Accordingly, the RTC is a target for clinically approved antiviral nucleoside analogs, including remdesivir 2 . Faithful synthesis of viral RNAs by the RTC requires recognition of the correct nucleotide triphosphate (NTP) for incorporation into the nascent RNA. To be effective inhibitors, antiviral nucleoside analogs must compete with the natural NTPs for incorporation. How the SARS-CoV-2 RTC discriminates between the natural NTPs, and how antiviral nucleoside analogs compete, has not been discerned in detail. Here, we use cryo-electron microscopy to visualize the RTC bound to each of the natural NTPs in states poised for incorporation. Furthermore, we investigate the RTC with the active metabolite of remdesivir, remdesivir triphosphate (RDV-TP), highlighting the structural basis for the selective incorporation of RDV-TP over its natural counterpart ATP 3,4 . Our results elucidate the suite of interactions required for NTP recognition, informing the rational design of antivirals. Our analysis also yields insights into nucleotide recognition by the nsp12 NiRAN, an enigmatic catalytic domain essential for viral propagation 5 . The NiRAN selectively binds GTP, strengthening proposals for the role of this domain in the formation of the 5’ RNA cap 6 .

The first step in gene expression in all organisms requires opening the DNA duplex to expose one strand for templated RNA synthesis. In Escherichia coli, promoter DNA sequence fundamentally determines how fast the RNA polymerase (RNAP) forms "open" complexes (RPo), whether RPo persists for seconds or hours, and how quickly RNAP transitions from initiation to elongation. These rates control promoter strength in vivo, but their structural origins remain largely unknown. Here, we use cryoelectron microscopy to determine the structures of RPo formed de novo at three promoters with widely differing lifetimes at 37 °C: λPR (t1/2 ∼10 h), T7A1 (t1/2 ∼4 min), and a point mutant in λPR (λPR-5C) (t1/2 ∼2 h). Two distinct RPo conformers are populated at λPR, likely representing productive and unproductive forms of RPo observed in solution studies. We find that changes in the sequence and length of DNA in the transcription bubble just upstream of the start site (+1) globally alter the network of DNA-RNAP interactions, base stacking, and strand order in the single-stranded DNA of the transcription bubble; these differences propagate beyond the bubble to upstream and downstream DNA. After expanding the transcription bubble by one base (T7A1), the nontemplate strand "scrunches" inside the active site cleft; the template strand bulges outside the cleft at the upstream edge of the bubble. The structures illustrate how limited sequence changes trigger global alterations in the transcription bubble that modulate the RPo lifetime and affect the subsequent steps of the transcription cycle.

Significance The modulation of the rate of formation and of the lifetime of transcription initiation complexes is a critical point in gene expression control. In Escherichia coli, single-nucleotide changes can change the half-life of an RNA polymerase (RNAP)–promoter DNA complex by more than an order of magnitude. The origins of these effects are poorly understood. Using cryoelectron microscopy, we find that small alterations in the sequence or size of the transcription bubble trigger global changes in RNAP–DNA interactions and in DNA base stacking. Our results reveal that nonadditive structural changes allow a few crucial DNA positions to tune the transcription initiation complex lifetime from seconds to hours, influencing the rate and efficiency of the initial steps of RNA synthesis. The first step in gene expression in all organisms requires opening the DNA duplex to expose one strand for templated RNA synthesis. In Escherichia coli, promoter DNA sequence fundamentally determines how fast the RNA polymerase (RNAP) forms “open” complexes (RPo), whether RPo persists for seconds or hours, and how quickly RNAP transitions from initiation to elongation. These rates control promoter strength in vivo, but their structural origins remain largely unknown. Here, we use cryoelectron microscopy to determine the structures of RPo formed de novo at three promoters with widely differing lifetimes at 37 °C: λPR (t1/2 ∼10 h), T7A1 (t1/2 ∼4 min), and a point mutant in λPR (λPR-5C) (t1/2 ∼2 h). Two distinct RPo conformers are populated at λPR, likely representing productive and unproductive forms of RPo observed in solution studies. We find that changes in the sequence and length of DNA in the transcription bubble just upstream of the start site (+1) globally alter the network of DNA–RNAP interactions, base stacking, and strand order in the single-stranded DNA of the transcription bubble; these differences propagate beyond the bubble to upstream and downstream DNA. After expanding the transcription bubble by one base (T7A1), the nontemplate strand “scrunches” inside the active site cleft; the template strand bulges outside the cleft at the upstream edge of the bubble. The structures illustrate how limited sequence changes trigger global alterations in the transcription bubble that modulate the RPo lifetime and affect the subsequent steps of the transcription cycle.

This paper provides an overview of the discussion and presentations from the Workshop on the Management of Large CryoEM Facilities held at the New York Structural Biology Center, New York, NY on February 6-7, 2017. A major objective of the workshop was to discuss best practices for managing cryoEM facilities. The discussions were largely focused on supporting single-particle methods for cryoEM and topics included: user access, assessing projects, workflow, sample handling, microscopy, data management and processing, and user training.

Defects in the mitochondrial electron transport chain (ETC) underlie the pathogenesis of many diseases ranging from metabolic syndrome to heart failure, and Alzheimer's Disease. Optimization of electron transfer (ET) through ETC provides a unifying therapeutic approach to these diverse disorders. SS‐31 is a synthetic tetrapeptide that selectively targets the inner mitochondrial membrane and has been shown to protect mitochondrial integrity, preserve ATP synthesis, and minimize ROS formation in numerous animal models. We now report that that SS‐31 acts by facilitating ET in the ETC. In isolated mitochondria, SS‐31 potentiated O2 consumption and ATP synthesis initiated by complex I or II substrates, or by direct reduction of cyt C. SS‐31 also promoted ET in cyt C‐deficient mitoplasts, suggesting that SS‐31 facilitates ET through cyt C, especially under rate‐limiting conditions. Circular dichroism and Trp fluorescence verified that SS‐31 interacts directly with cyt C. Photoluminescence and cyclic voltammetry established that SS‐31 increases electron capacity of cyt C and promotes electron diffusion through cyt C. We conclude that SS‐31 minimized the energy barrier for ET through cyt C. This concept represents a novel approach for protecting cellular energy and a paradigm shift in the treatment of a multitude of diseases associated with mitochondrial dysfunction.