Structural consequences of deproteinating the 50S ribosome

Abstract

ABSTRACTRibosomes are complex ribonucleoprotein particles. Purified 50S ribosomes subjected to high-salt wash, removing a subset of ribosomal proteins (r-proteins), were early shown competent for in vitro assembly into functional 50S subunits. We here used cryo-EM to determine the structure of such LiCl core particles derived from E. coli 50S subunits. A wide range of complexes with large variation in extent of ordered 23S rRNA and occupancy of r-proteins could be identified, and resolved to between 2.8 Å and 9 Å resolution. Many of these particles showed high similarity to in vivo and in vitro assembly intermediates, supporting the inherent stability or metastability of these states. Similar to states in early ribosome assembly, the main class showed ordered density for 23S rRNA domains 0, I, II, III, VI and the 5’-half of domain IV. In addition, smaller core particles were discovered, which show that the most stable part of the 50S under high-salt conditions includes parts of domain 0 and most of domains I, III and the 5’-half of domain IV and four to eight r-proteins. Our data support a multi-pathway disassembly process based on independent folding blocks, similar but reverse to the assembly process. The study provides examples of dependencies between complex tertiary RNA structure and RNA-protein interactions where protein extensions dissociate before the globular domains. We observe formation of a non-native RNA structure upon protein dissociation, demonstrating that r-proteins stabilize native RNA structure and prevent non-native interactions also after folding.IMPORTANCERibosome assembly and stability remain only partially understood. Incubation of ribosomes with salts was early shown to induce dissociation of the more loosely bound ribosomal proteins (r-proteins) and formation of so-called core particles. In this work, cryo-EM imaging of 50S LiCl core particles from E. coli for the first time allowed structural characterization of such particles of different size. The smallest particles demonstrate what constitutes the smallest stable core of the 50S ribosomal subunit, and the sequential comparison with larger particles show how the ribosome disassembles and assembles in layers of rRNA structure stabilized by globular domains and extended tails of r-proteins. Major insights are that ribosomes disassemble along different paths, that dissociation of r-proteins can induce misfolding of rRNA and that extended tails of r-proteins dissociate from rRNA before the globular domains. The characterized particles can be used in future mechanistic studies of ribosome biogenesis.