RNA polymerase III (Pol III), the largest eukaryote polymerase yet characterized, transcribes structured small non-coding RNAs; here cryo-electron microscopy structures of budding yeast Pol III allow building of an atomic-level model of the complete 17-subunit complex, both unbound and while elongating RNA. Transcription of genes encoding small structured RNAs such as transfer RNAs, spliceosomal U6 small nuclear RNA and ribosomal 5S RNA is carried out by RNA polymerase III (Pol III), the largest yet structurally least characterized eukaryotic RNA polymerase. Here we present the cryo-electron microscopy structures of the Saccharomyces cerevisiae Pol III elongating complex at 3.9 Å resolution and the apo Pol III enzyme in two different conformations at 4.6 and 4.7 Å resolution, respectively, which allow the building of a 17-subunit atomic model of Pol III. The reconstructions reveal the precise orientation of the C82–C34–C31 heterotrimer in close proximity to the stalk. The C53–C37 heterodimer positions residues involved in transcription termination close to the non-template DNA strand. In the apo Pol III structures, the stalk adopts different orientations coupled with closed and open conformations of the clamp. Our results provide novel insights into Pol III-specific transcription and the adaptation of Pol III towards its small transcriptional targets. RNA polymerase III (Pol III) transcribes structured small non-coding RNAs such as tRNAs and spliceosomal RNAs. It is the largest eukaryote polymerase, yet the least characterized structurally. Here Christoph Müller and colleagues determine the cryo-electron microscopy structures of budding yeast Pol III and build an atomic-level model of the complete 17-subunit complex, in both unbound and elongating conformations. The results allow a detailed comparison with the Pol I and Pol II enzymes and explain the adaptation of this polymerase towards its specific targets.

Protein S-persulfidation (P-SSH) is recognized as a common posttranslational modification. It occurs under basal conditions and is often observed to be elevated under stress conditions. However, the mechanism(s) by which proteins are persulfidated inside cells have remained unclear. Here we report that 3-mercaptopyruvate sulfur transferase (MPST) engages in direct protein-to-protein transpersulfidation reactions beyond its previously known protein substrates thioredoxin and MOCS3/Uba4, associated with H₂S generation and transfer RNA thiolation, respectively. We observe that depletion of MPST in human cells lowers overall intracellular protein persulfidation levels and identify a subset of proteins whose persulfidation depends on MPST. The predicted involvement of these proteins in the adaptation to stress responses supports the notion that MPST-dependent protein persulfidation promotes cytoprotective functions. The observation of MPST-independent protein persulfidation suggests that other protein persulfidases remain to be identified.

In many animals, the germ plasm segregates germline from soma during early development. Oskar protein is known for its ability to induce germ plasm formation and germ cells in Drosophila. However, the molecular basis of germ plasm formation remains unclear. Here, we show that Oskar is an RNA-binding protein in vivo, crosslinking to nanos, polar granule component, and germ cell-less mRNAs, each of which has a role in germline formation. Furthermore, we present high-resolution crystal structures of the two Oskar domains. RNA-binding maps in vitro to the C-terminal domain, which shows structural similarity to SGNH hydrolases. The highly conserved N-terminal LOTUS domain forms dimers and mediates Oskar interaction with the germline-specific RNA helicase Vasa in vitro. Our findings suggest a dual function of Oskar in RNA and Vasa binding, providing molecular clues to its germ plasm function.

The highly conserved eukaryotic Elongator complex performs specific chemical modifications on wobble base uridines of tRNAs, which are essential for proteome stability and homeostasis. The complex is formed by six individual subunits (Elp1‐6) that are all equally important for its tRNA modification activity. However, its overall architecture and the detailed reaction mechanism remain elusive. Here, we report the structures of the fully assembled yeast Elongator and the Elp123 sub‐complex solved by an integrative structure determination approach showing that two copies of the Elp1, Elp2, and Elp3 subunits form a two‐lobed scaffold, which binds Elp456 asymmetrically. Our topological models are consistent with previous studies on individual subunits and further validated by complementary biochemical analyses. Our study provides a structural framework on how the tRNA modification activity is carried out by Elongator.