Concurrent nucleation of 16S folding and induced fit in 30S ribosome assembly

Abstract

SummaryRapidly growing cells produce thousands of new ribosomes each minute, in a tightly regulated process that is essential to cell growth. 1,2 How the 16S rRNA and 20 proteins that make up the 30S ribosomal subunit assemble faithfully in a few minutes remains a challenging problem, in part because real-time data on the earliest stages of assembly are lacking. Here, we show that 30S assembly nucleates concurrently from different points along the rRNA, by providing snapshots of individual RNA and protein interactions as they emerge in real time. Time-resolved hydroxyl radical footprinting 3 was used to map changes in the structure of the rRNA within 20 ms after addition of total 30S proteins. Helix junctions in each domain fold within 100 ms. By contrast, interactions surrounding the decoding site and between the 5′, central and 3′ domains require 2-200 seconds to form. Surprisingly, nucleotides contacted by the same protein are protected at different rates, indicating that initial RNA-protein encounter complexes refold during assembly. While early steps in assembly are linked to intrinsically stable rRNA structure, later steps correspond to regions of induced fit between the proteins and the rRNA.Nomura and colleagues demonstrated that hierarchical addition of ribosomal proteins to the 16S rRNA produces cooperativity, 4 that is due to protein-induced structural changes in the 16S rRNA rather than direct contacts between proteins. 5 As the rRNA becomes more structured as proteins join the complex, assembly is coupled to the folding pathway of the rRNA. 6,7 The simplest kinetic model for 30S assembly is that regions of the 16S rRNA contacted by primary assembly proteins fold earliest, while helices stabilized by tertiary assembly proteins fold last. If assembly is strictly sequential, each subdomain of the rRNA will fold within a distinct time, producing a limited set of intermediate complexes.Alternatively, the rate of protein binding may initially depend on the stochastic probability of forming locally stable rRNA and protein interactions, with progression to later intermediates depending on propagation of this conformational order to neighboring regions in the rRNA. If more than one region of the naked rRNA can fold, assembly is expected to nucleate from many places at once, producing an ensemble of reconstitution intermediates and multi-stage assembly kinetics. 8 *Corresponding author: E-mail: swoodson@jhu.edu, tel. +001-410-516-2015, FAX +001-410-516-4118 To visualize the intermediates of 30S ribosome assembly, the structure of the 16S rRNA was probed by time-resolved X-ray hydroxyl radical footprinting (Figure 1). The extent of RNA cleavage correlates with backbone exposure. 9 Thus, this method probes individual tertiary contacts in the rRNA as well as protein interactions that bury the rRNA backbone. Previous efforts to map the conformational changes in the 16S rRNA during 30S ribosome assembly used low temperature or subsets of proteins to stall assembly at specific stages. 10,11 We took advantage of the...

A hierarchical model for assembly of eukaryotic 60S ribosomal subunit domains

“…Furthermore, we also know that most RPLs are found in the earliest pre-60S particles, yet their association with preribosomes is thought to become more stable as assembly proceeds, by creating more contacts with pre-rRNA (Ferreira-Cerca et al 2007;Adilakshmi et al 2008;Ohmayer et al 2013). Thus, analyzing the RPL content of preribosomes depleted of early-, middle-, and late-acting RPLs enabled us to arrive at a conclusion that assembly of the 60S ribosomal subunit is hierarchical.…”

“…Most RPs assemble with preribosomes early in the biogenesis pathway (Kruiswijk et al 1978;Ferreira-Cerca et al 2007;Babiano and de la Cruz 2010;Babiano et al 2012;Gamalinda et al 2013;Ohmayer et al 2013). As the nascent subunits mature, the association of RPs with pre-rRNA is strengthened by dynamic rearrangement of initial protein-RNA encounter complexes (Ferreira-Cerca et al 2007;Adilakshmi et al 2008;Ohmayer et al 2013).…”

“…Their binding induces conformational changes in rRNA, which enables the subsequent assembly of secondary and tertiary binding RPs (Shajani et al 2011;Woodson 2011). More recent studies have shown that assembly in vitro can proceed via parallel pathways (Adilakshmi et al 2008;Mulder et al 2010) in which individual RPs contact different rRNA elements in multiple stages throughout assembly.…”

Quantitation of ten 30S ribosomal assembly intermediates using fluorescence triple correlation spectroscopy

“…The Nomura maps documented how the omission of one protein precludes incorporation of others, but only monitored stable protein-RNA interactions that would survive the biochemical purification steps including nonequilibrium ultracentrifugation. Recent RNA protection data suggest that weak protein-RNA interactions form rapidly during assembly (5,6), raising the question of how completely the map represents the types of intermediates that form during assembly.…”

“…Approximate rate constants and orders of binding are known from kinetic experiments, but kinetic profiles for individual protein binding and RNA folding steps are complex and defy simple models (1)(2)(3)(4)(5)(6)(7)(8). The fundamentally kinetic process of ribosome assembly is thus still best understood in terms of the thermodynamic (9) maps generated by the Nomura group in the 1970s (10).…”