Force measurements show that uL4 and uL24 mechanically stabilize a fragment of 23S rRNA essential for ribosome assembly

Abstract

In vitro reconstitution studies have shown that ribosome assembly is highly cooperative and starts with the binding of a few ribosomal (r-) proteins to rRNA. It is unknown how these early binders act. Focusing on the initial stage of the assembly of the large subunit of the Escherichia coli ribosome, we prepared a 79-nucleotide-long region of 23S rRNA encompassing the binding sites of the early binders uL4 and uL24. Force signals were measured in a DNA/RNA dumbbell configuration with a double optical tweezers setup. The rRNA fragment was stretched until unfolded, in the absence or in the presence of the r-proteins (either uL4, uL24, or both). We show that the r-proteins uL4 and uL24 individually stabilize the rRNA fragment, both acting as molecular clamps. Interestingly, this mechanical stabilization is enhanced when both proteins are bound simultaneously. Independently, we observe a cooperative binding of uL4 and uL24 to the rRNA fragment. These two aspects of r-proteins binding both contribute to the efficient stabilization of the 3D structure of the rRNA fragment under investigation. We finally consider implications of our results for large ribosomal subunit assembly.

Keywords: optical trap; ribosome assembly; single molecule.

FIGURE 1.

Typical force versus displacement curves. (A) A dual optical trap is used to manipulate a DNA/RNA molecular construction in a dumbbell configuration. In this configuration, the RNA containing the E. coli 23S rRNA fragment of interest (from nucleotides 281 to 359—secondary structure represented schematically) is hybridized to two DNA handles both containing a biotin moiety (orange) at one of their extremities. Biotin moieties are linked to streptavidin (red) coated silica beads. (B) Typical bare rRNA force versus displacement curve. As the molecular construction is stretched, one observes a sawtooth signal region around 10 pN (marked by the black arrow) corresponding to the progressive unfolding of the rRNA fragment of interest. Unfolding (green) and refolding (light green) curves as indicated by the corresponding arrows have been plotted showing little hysteresis. (Inset) A close-up shows the unfolding experimental sawtooth signal (green) and the idealized path (black) around 10 pN. (C) Region corresponding to the unfolding of the rRNA fragment of interest. A typical curve is represented for each set of experiments: bare rRNA fragment (green), rRNA fragment bound to uL4 (blue), rRNA fragment bound to uL24 (brown), rRNA fragment bound to both uL4 and uL24 (red). The curves have been shifted horizontally for better visualization. Arrows indicate most important force drops. In black are superimposed the idealized paths extracted from the analysis of the force versus displacement curves. These paths allow the precise assignation of the observed intermediate states during the progressive unfolding of the rRNA fragment of interest.

FIGURE 2.

(A) Superposition of the idealized paths of all the measurements performed without r-proteins (254 measurements). All the curves have been superimposed in a heat map histogram where red (dark blue) indicates frequently (rarely) observed states. (B) Superposition of the idealized paths of all the measurements performed with 8 µM of each r-protein uL4 and uL24 (255 measurements). (C) Secondary structure of the rRNA fragment. The colored bars (areas) indicate the mean position (standard deviation) of the two RNA unfolding intermediate states I₁ and I₃ located in H18. (D) Secondary structure of the rRNA fragment of interest showing nucleotides involved in RNA/uL24 (brown) and RNA/uL4 (blue) contacts observed in the fully assembled 50S ribosomal subunit (analyzed from PDB entry 4YBB).

FIGURE 3.

Histograms of the maximum force reached by states of released length below 25 nm (region corresponding to F, I₁, and I₃) for experiments performed (A) without r-protein (254 measurements), (B) with uL4 at 8 µM (45 measurements), (C) with uL24 at 15 µM (65 measurements), (D) with both uL4 and uL24 at 8 µM each (255 measurements). Each bar represents the fraction of curves where the maximum force reached by the intermediates occurs in the corresponding force range (with standard errors pictured as error bars). To determine the typical force necessary to unfold each possible rRNA fragment-protein complex, each distribution of forces displayed in panels A–D has been fitted as the sum of gaussians (the procedure is thoroughly described in our Supplemental Material paragraph “Estimation of the typical force involved in the mechanical stabilization”). In panel A, where there is no protein, the distribution of forces has been fitted using a single Gaussian (green curve) and the characteristic force is displayed in green. This gaussian is used subsequently for panels B–D. In panels B and C, where only one of the two proteins is present in solution, each distribution is the sum of two gaussians (one displayed in green: no protein bound [previously determined]; and one in blue or brown [uL4- or uL24-bound RNA fragment, respectively]). In panel D (the two proteins in solution), the distribution is fitted by a model which is the sum of four gaussians, corresponding to the four possible cases: no protein, uL4 bound, uL24 bound, uL4 + uL24 bound. The first three of them were previously determined. The fourth gaussian is newly determined and shown in red. In panels B–D, the sum of the gaussians is represented as a dashed line and thus corresponds to the best fit to the complete data. The mean force of each gaussian is indicated.

FIGURE 4.

Minimal cooperative binding scheme of r-proteins uL4 and uL24 to the rRNA fragment: Association constants directly measured from our data are indicated (i.e., 0.06 ± 0.01 µM⁻¹ for uL24; 1.5 ± 0.2 µM⁻¹ for uL4; 0.33 ± 0.01 µM⁻¹ for uL24 when uL4 is already bound to the rRNA fragment; 10.6 ± 3.5 µM⁻¹ for uL4 when uL24 is already bound to the rRNA fragment; all figures correspond to mean ± standard error).

FIGURE 5.

Ribbon representation of the 3D structure of the RNA and proteins investigated in this paper—structure as it is observed in the fully assembled 50S subunit (PDB entry 4YBB). The RNA is colored following the conventions shown in the inset; the r-proteins uL4 and uL24 are pictured in blue and orange respectively. (Inset) Color conventions used to highlight the H18/H19/H20 three-way junction: top, 2D representation of the junction; bottom, schematic representation of the 3D structure of the junction—a prototypic Y-shaped three-way junction, as described in the text. In this picture are also shown schematically the tertiary interactions that stabilize this particular 3D structure: RNA–RNA interactions (black dots) and protein–RNA interactions (arrows).