Visualizing ribosome biogenesis: parallel assembly pathways for the 30S subunit

Abstract

Ribosomes are self-assembling macromolecular machines that translate DNA into proteins, and an understanding of ribosome biogenesis is central to cellular physiology. Previous studies on the Escherichia coli 30S subunit suggest that ribosome assembly occurs via multiple parallel pathways rather than through a single rate-limiting step, but little mechanistic information is known about this process. Discovery single-particle profiling (DSP), an application of time-resolved electron microscopy, was used to obtain more than 1 million snapshots of assembling 30S subunits, identify and visualize the structures of 14 assembly intermediates, and monitor the population flux of these intermediates over time. DSP results were integrated with mass spectrometry data to construct the first ribosome-assembly mechanism that incorporates binding dependencies, rate constants, and structural characterization of populated intermediates.

Figure 1. Discovery Single-particle Profiling (DSP)

(A) Assembly of the 30S subunit was initiated as described (9), and aliquots were prepared for negative stain EM (16) at various time points. The DSP method was used to discover sub populations of assembly intermediates from a merged time course dataset of a single assembly reaction (see Supplementary Discussion). (B) Each row of the array results from a single time point, and particle averages are ranked from left to right by decreasing population with a heat map overlaid according to percentage of particles that populate a given class; red: 8–11%, yellow: 6–8%, green: 4–6%, blue: 2–4%.

Figure 2. Structural Determination of Assembly Intermediates

Assembly intermediates classify into four groups based on classification of 2D data (Figures S2–3,S5), docking of the 30S subunit crystal structure (Figure S8B), and 3D difference mapping (Figure S6). RCT volumes are color coded in accordance with state of assembly where red < green < blue < purple. A conformational change in the neck of central domain rRNA causing a rotation in the head domain is the major difference between Group II and Group IV intermediates (Δ and arrow).

Figure 3. Assembly Intermediate Population Flux over Time

(A) Classification of merged time point datasets allowed determination of the contribution of different time points to assembly intermediate groups I (red), II (green), III (blue) and IV (purple). (B) Measurement of protein binding rates via PC-QMS illustrates the correspondence between population profiles determined via DSP and in vitro 30S subunit assembly kinetics. (C) Decomposition of population flux for individual assembly intermediates within group IV (purple) revealed rapid accumulation of S2-containing assembly populations, suggesting the existence of an S2-mediated kinetic trap in the assembly mechanism.

Figure 4. A Parallel Mechanism for 30S Subunit Assembly

(A) A Nomura assembly map on a time axis that takes into account the t_0.5 of binding for each r-protein as determined via PC-QMS. (B) Combination of kinetic, thermodynamic, and single-particle data allows construction of a mechanism for 30S subunit assembly that accounts for parallel assembly pathways.