Structures of the simplest replisome: The DNA replisome performs concerted parental-strand separation and DNA synthesis on both strands. Gao et al. report the cryo–electron microscopy structures of the minimum set of bacteriophage T7 proteins that can carry out leading- and lagging-strand synthesis at the replication fork (see the Perspective by Li and O'Donnell). Three key enzymes involved in DNA replication—DNA polymerase, helicase, and primase—were visualized in complex with substrate DNA, demonstrating their highly dynamic organizations on both strands. Comparison of prokaryotic and eukaryotic replisomes reveals evolutionarily conserved operating principles and provides a structural basis for understanding coordination among DNA replication, recombination, and repair. Science , this issue p. eaav7003 ; see also p. 814

Understanding splicing from the 3′ end: The spliceosome removes introns from eukaryotic mRNA precursors and yields mature transcripts by joining exons. Despite decades of functional studies and recent progress in understanding the spliceosome structure, the mechanism by which the 3′ splice site (SS) is recognized by the spliceosome has remained unclear. Liu et al. and Wilkinson et al. report the high-resolution cryo-electron microscopy structures of the yeast postcatalytic spliceosome. The structures reveal that the 3′SS is recognized through non-Watson-Crick base pairing with the 5′SS and the branch point, stabilized by the intron region and protein factors. Science , this issue p. 1278 , p. 1283

ClC-1 protein channels facilitate rapid passage of chloride ions across cellular membranes, thereby orchestrating skeletal muscle excitability. Malfunction of ClC-1 is associated with myotonia congenita, a disease impairing muscle relaxation. Here, we present the cryo-electron microscopy (cryo-EM) structure of human ClC-1, uncovering an architecture reminiscent of that of bovine ClC-K and CLC transporters. The chloride conducting pathway exhibits distinct features, including a central glutamate residue (“fast gate”) known to confer voltage-dependence (a mechanistic feature not present in ClC-K), linked to a somewhat rearranged central tyrosine and a narrower aperture of the pore toward the extracellular vestibule. These characteristics agree with the lower chloride flux of ClC-1 compared with ClC-K and enable us to propose a model for chloride passage in voltage-dependent CLC channels. Comparison of structures derived from protein studied in different experimental conditions supports the notion that pH and adenine nucleotides regulate ClC-1 through interactions between the so-called cystathionine-β-synthase (CBS) domains and the intracellular vestibule (“slow gating”). The structure also provides a framework for analysis of mutations causing myotonia congenita and reveals a striking correlation between mutated residues and the phenotypic effect on voltage gating, opening avenues for rational design of therapies against ClC-1–related diseases. Chloride transporting CLC proteins are expressed in a wide range of organisms, and the family encompasses several members with numerous roles in human health and disease by allowing movement of chloride ions across the membranes that encapsulate cells and cellular organelles. Structurally, CLCs form dimers possessing a separate ion translocation pathway in each monomer, and they can operate as either channels or transporters that exchange chloride for protons. The CLC channel ClC-1 is critical to skeletal muscle excitability and has been proposed as a target to alleviate neuromuscular disorders. Here, we have analyzed the structure of human ClC-1 and revealed the high similarity of its ion conducting pathway to those observed in other CLC members, including prokaryotic and algal transporters. Our data suggest how ClC-1 is regulated by environmental cues to allow opening and closure, thereby permitting attenuation of muscle function. Our results help with understanding the principal determinants that govern CLC proteins and may guide downstream translational applications to combat muscle pathologies.

We report the asymmetric reconstruction of the single-stranded RNA (ssRNA) content in one of the three otherwise identical virions of a multipartite RNA virus, brome mosaic virus (BMV). We exploit a sample consisting exclusively of particles with the same RNA content—specifically, RNAs 3 and 4—assembled in planta by agrobacterium-mediated transient expression. We find that the interior of the particle is nearly empty, with most of the RNA genome situated at the capsid shell. However, this density is disordered in the sense that the RNA is not associated with any particular structure but rather, with an ensemble of secondary/tertiary structures that interact with the capsid protein. Our results illustrate a fundamental difference between the ssRNA organization in the multipartite BMV viral capsid and the monopartite bacteriophages MS2 and Qβ for which a dominant RNA conformation is found inside the assembled viral capsids, with RNA density conserved even at the center of the particle. This can be understood in the context of the differing demands on their respective lifecycles: BMV must package separately each of several different RNA molecules and has been shown to replicate and package them in isolated, membrane-bound, cytoplasmic complexes, whereas the bacteriophages exploit sequence-specific “packaging signals” throughout the viral RNA to package their monopartite genomes.