RUVBL1-RUVBL2 AAA-ATPase: a versatile scaffold for multiple complexes and functions.

RUVBL1 and RUVBL2 are two highly conserved AAA+ ATPases that form a hetero-hexameric complex that participates in a wide range of unrelated cellular processes, including chromatin remodeling, Fanconi Anemia (FA), nonsense-mediated mRNA decay (NMD), and assembly and maturation of several large macromolecular complexes such as RNA polymerases, the box C/D small nucleolar ribonucleoprotein (snoRNP) and mTOR complexes. How the RUVBL1-RUVBL2 complex works in such a variety of processes, sometimes antagonistic, has been obscure for a long time. Recent cryo-electron microscopy (cryo-EM) studies have started to reveal how RUVBL1-RUVBL2 forms a scaffold for complex protein-protein interactions and how the structure and ATPase activity of RUVBL1-RUVBL2 can be affected and regulated by the interaction with clients.

Molecular basis of tRNA recognition by the Elongator complex.

The highly conserved Elongator complex modifies transfer RNAs (tRNAs) in their wobble base position, thereby regulating protein synthesis and ensuring proteome stability. The precise mechanisms of tRNA recognition and its modification reaction remain elusive. Here, we show cryo-electron microscopy structures of the catalytic subcomplex of Elongator and its tRNA-bound state at resolutions of 3.3 and 4.4 Å. The structures resolve details of the catalytic site, including the substrate tRNA, the iron-sulfur cluster, and a SAM molecule, which are all validated by mutational analyses in vitro and in vivo. tRNA binding induces conformational rearrangements, which precisely position the targeted anticodon base in the active site. Our results provide the molecular basis for substrate recognition of Elongator, essential to understand its cellular function and role in neurodegenerative diseases and cancer.

Structural asymmetry in the eukaryotic Elongator complex.

Nucleoside modifications in tRNA anticodons regulate ribosome dynamics during translation elongation and, thereby, fine-tune global protein synthesis rates. The highly conserved eukaryotic Elongator complex conducts specific C5-substitutions in tRNA wobble base uridines. It harbors two copies of each of its six individual subunits, which are all equally important for its activity. Here, we summarize recent developments focusing on the architecture of the Elongator complex, showing an asymmetric subunit arrangement, and its functional implications. In addition, we discuss the role of its proposed active site, its individual subunits and temporarily associated regulatory factors. Finally, we aim to provide mechanistic explanations for the link between mutations in Elongator subunits and the onset of several severe human pathologies.

Architecture of the yeast Elongator complex.

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.

The Combination of X-Ray Crystallography and Cryo-Electron Microscopy Provides Insight into the Overall Architecture of the Dodecameric Rvb1/Rvb2 Complex.

The Rvb1/Rvb2 complex is an essential component of many cellular pathways. The Rvb1/Rvb2 complex forms a dodecameric assembly where six copies of each subunit form two heterohexameric rings. However, due to conformational variability, the way the two rings pack together is still not fully understood. Here, we present the crystal structure and two cryo-electron microscopy reconstructions of the dodecameric, full-length Rvb1/Rvb2 complex, all showing that the interaction between the two heterohexameric rings is mediated through the Rvb1/Rvb2-specific domain II. Two conformations of the Rvb1/Rvb2 dodecamer are present in solution: a stretched conformation also present in the crystal, and a compact conformation. Novel asymmetric features observed in the reconstruction of the compact conformation provide additional insight into the plasticity of the Rvb1/Rvb2 complex.

Nucleic acid packaging in viruses.

Viruses protect their genetic information by enclosing the viral nucleic acid inside a protein shell (capsid), in a process known as genome packaging. Viruses follow essentially two main strategies to package their genome: Either they co-assemble their genetic material together with the capsid protein, or they assemble first an empty shell (procapsid) and then pump the genome inside the capsid with a molecular motor that uses the energy released by ATP hydrolysis. During packaging the viral nucleic acid is condensed to very high concentration by its careful arrangement in concentric layers inside the capsid. In this chapter we will first give an overview of the different strategies used for genome packaging to discuss later some specific virus models where the structures of the main proteins involved, and the biophysics underlying the packaging mechanism, have been well documented.

Large terminase conformational change induced by connector binding in bacteriophage T7.

During bacteriophage morphogenesis DNA is translocated into a preformed prohead by the complex formed by the portal protein, or connector, plus the terminase, which are located at an especial prohead vertex. The terminase is a powerful motor that converts ATP hydrolysis into mechanical movement of the DNA. Here, we have determined the structure of the T7 large terminase by electron microscopy. The five terminase subunits assemble in a toroid that encloses a channel wide enough to accommodate dsDNA. The structure of the complete connector-terminase complex is also reported, revealing the coupling between the terminase and the connector forming a continuous channel. The structure of the terminase assembled into the complex showed a different conformation when compared with the isolated terminase pentamer. To understand in molecular terms the terminase morphological change, we generated the terminase atomic model based on the crystallographic structure of its phage T4 counterpart. The docking of the threaded model in both terminase conformations showed that the transition between the two states can be achieved by rigid body subunit rotation in the pentameric assembly. The existence of two terminase conformations and its possible relation to the sequential DNA translocation may shed light into the molecular bases of the packaging mechanism of bacteriophage T7.

Stepwise motion of a microcantilever driven by the hydrolysis of viral ATPases.

The biomolecular machines involved in DNA packaging by viruses generate one of the highest mechanical powers observed in nature. One component of the DNA packaging machinery, called the terminase, has been proposed as the molecular motor that converts chemical energy from ATP hydrolysis into mechanical movement of DNA during bacteriophage morphogenesis. However, the conformational changes involved in this energy conversion have never been observed. Here we report a real-time measurement of ATP-induced conformational changes in the terminase of bacteriophage T7 (gp19). The recording of the cantilever bending during its functionalization shows the existence of a gp19 monolayer arrangement confirmed by atomic force microscopy of the immobilized proteins. The ATP hydrolysis of the gp19 terminase generates a stepped motion of the cantilever and points to a mechanical cooperative effect among gp19 oligomers. Furthermore, the effect of ATP can be counteracted by non-hydrolyzable nucleotide analogs.