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

Abstract

Rapidly growing cells produce thousands of new ribosomes each minute, in a tightly regulated process that is essential to cell growth. How the Escherichia coli 16S ribosomal RNA and the 20 proteins that make up the 30S ribosomal subunit can assemble correctly in a few minutes remains a challenging problem, partly because of the lack of real-time data on the earliest stages of assembly. By providing snapshots of individual RNA and protein interactions as they emerge in real time, here we show that 30S assembly nucleates concurrently from different points along the rRNA. Time-resolved hydroxyl radical footprinting was used to map changes in the structure of the rRNA within 20 milliseconds after the addition of total 30S proteins. Helical junctions in each domain fold within 100 ms. In contrast, interactions surrounding the decoding site and between the 5', the central and the 3' domains require 2-200 seconds to form. Unexpectedly, nucleotides contacted by the same protein are protected at different rates, indicating that initial RNA-protein encounter complexes refold during assembly. Although 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.

Figure 1. Time-resolved X-ray footprinting of 30S ribosome assembly

a, Native 16S rRNA and total protein from 30S subunits (TP30) were mixed in 5–10 ms and irradiated with a synchrotron X-ray beam, to cleave the RNA at exposed riboses. b, 16S fragments were analyzed by primer extension. Cleavage pattern 0.02 to 180 s post-TP30. RNA, pre-folded 16S rRNA; 30S, triplicate controls on native 30S subunits; UCAG, dideoxy sequence ladders; –, untreated RNA. Primer anneals after nt 1257. c, Relative saturation (Y) of each protection vs. assembly time, fit to single or double exponential rate equations (Supplemental Methods). (●), nt 398; (▲), nt 263; (■), nt 617–618; (○,+), average of 30S controls. Additional data are in Figure S3 and Table S1.

Figure 2. Simultaneous folding of 16S domains

a, Protected nucleotides (740 positions) were clustered according to the rate (time) constant for backbone protection and colored as indicated in the key. Where the amplitude of the initial burst phase is ≤ 60%, the slower rate constant is used. Grey (120 positions), rate constant undetermined due to a pause in reverse transcription or weak protection. Natural adenine methylation hampered quantitative analysis of residues in the decoding site. Circles, RNA-RNA contact; square, RNA-protein contact; solid symbols, buried C4′ in crystal structures, open circles, predicted C4′ ASA > 4 Ų. b, 3D ribbon of E. coli 16S rRNA (2awy) colored as in a, viewed from the 50S interface. See Figure S4 for additional views of each domain.

Figure 3. Stepwise assembly of RNA and protein interactions

a, Protein S20 (yellow ribbon) contacts the 30S body (5′ domain; grey) earlier than helix 44 in the 3′ minor domain (pink). 16S nts colored as in Figure 2. b, Proteins S7 (yellow) and S9 (green) protect a segment of their binding site immediately (red), while nucleotides at the interface between the subdomains are protected slowly (blue).

Figure 4. Ribosomal proteins interact with the rRNA in stages

a, b, Kinetics of direct rRNA backbone protection by protein S4 (magenta) and S7 (yellow), colored as in Figure 2. Schematic symbols also as in Figure 2. c, d, Progress curves for protection of individual residues in contact with S4 and S7; for clarity only fitted curves are shown (see data in Figure S5). See Supplemental Methods for definition of RNA-protein contacts.