A steric block in translation caused by the antibiotic spectinomycin

Abstract

The widely used antibiotic spectinomycin inhibits bacterial protein synthesis by blocking translocation of messenger RNA and transfer RNAs on the ribosome. Here, we show that in crystals of the Escherichia coli 70S ribosome spectinomycin binding traps a distinct swiveling state of the head domain of the small ribosomal subunit. Spectinomycin also alters the rate and completeness of reverse translocation in vitro. These structural and biochemical data indicate that in solution spectinomycin sterically blocks swiveling of the head domain of the small ribosomal subunit and thereby disrupts the translocation cycle.

Figure 1

Structure of spectinomycin and its interactions with the ribosome. a) Chemical structures of antibiotics spectinomycin (Spc) and neomycin (Neo). b) Overview of the binding sites of spectinomycin and neomycin in the context of the 70S ribosome. Spectinomycin bound to h34 is colored gold, and neomycins bound to h44 and H69 are colored red and green, respectively. The 16S rRNA, 23S rRNA, and 5S rRNA are shown in light blue, gray, and purple, respectively. Proteins of the small and large subunits are shown in dark blue and magenta, respectively. c) Difference Fourier (F_obs − F_obs) electron density map of spectinomycin bound to h34 of ribosome II. Observed amplitudes for the unliganded 70S ribosome structure (13) served as a reference. Positive (blue) and negative (red) difference densities are contoured at 3 SD from the mean.

Figure 2

Conformational changes in the position of the head domain of the 30S subunit induced by spectinomycin. a, b) Difference Fourier (F_obs − F_obs) electron density map in the context of the 30S ribosomal subunit (panel a) and in the proximity of the spectinomycin binding site in h34 (panel b, stereoview) of ribosome II. Positive (blue) and negative (red) difference densities are contoured at 3 SD from the mean. 16S rRNA is in light blue, and small subunit proteins are in dark blue. Spectinomycin is colored gold. For panel a, the direction of the view is indicated by the ribosome icon. c) Superposition of spectinomycin-bound (Spc) and unliganded (II) (13) conformations of ribosome II in the 70S ribosome crystals. The rRNA is shown in red and gray, and proteins are purple and gray for spectinomycin-bound (Spc) and unliganded (II) (13) conformations of ribo-some II, respectively. d) Superposition of 30S subunits within the 70S ribosome. Differences in the position of phosphorous atoms in 16S rRNA (light colors) and Cα positions in proteins S7, S13, and S19 (darker colors) in the 30S subunit head domain are shown as vectors, as follows. Vectors from apo-ribosome II (13) to spectinomycin-bound ribosome II, red; vectors from spectinomycin-bound ribosome II to spectinomycin-bound ribosome I, blue. The 50S subunit from ribosome I (13) is shown in gray (23S rRNA), purple (5S rRNA), and magenta (large subunit proteins). The direction of the conformational change induced by spectinomycin is indicated by the red arrow. The path tRNAs travel during translocation from the A site to the E site is indicated by the black arrow. The direction of the view is indicated by the ribosome icon.

Figure 3

Effect of spectinomycin on reverse translocation. a) Schematics of the stepwise experimental setup for the EF-G dependent forward translocation (top) and spontaneous reverse translocation (bottom) reactions. The steps and order of antibiotic addition are described in Methods and are not depicted in the figure. b) Reverse translocation promoted by antibiotics in the presence of EF-G and GTP. The P complex corresponds to deacylated tRNA^fMet bound to the P site of the ribosome; the A complex (PRE) forms upon addition of the N-acetyl-Val-tRNA^Val to the A site of the P complex; G complex (POST) forms upon addition of EF-G/GTP to the A complex; the other complexes are formed upon subsequent addition of neomycin (Neo), spectinomycin (Spc), both neomycin and spectinomycin (Neo/Spc), or water (–) to the G (POST) complex. The percent of POST complex remaining at the end of the incubation is indicated below each lane. Reported values represent the mean values (±SEM) derived from three independent experiments. c) Spontaneous reverse translocation upon addition of E-site tRNA to ribosomes programmed with mRNA and P-site tRNA. Ribosomes were preincubated with spectinomycin prior to E-site tRNA addition in reactions involving spectinomycin (filled symbols). In reactions involving neomycin, neomycin was added simultaneously with E-site tRNA. Each experiment was carried out in triplicate. Kinetic parameters derived from the fitting of the data are reported in Table 1.

Figure 4

Conformational changes in the mRNA downstream tunnel that accompany mRNA unwinding and movement through the ribosome, viewed from the solvent side of the 30S subunit. The mRNA from ref 46 superimposed with the ribosome structures (13, 47) is shown in red. The 16S rRNA is shown in light blue, and proteins S3, S4, and S5 are dark blue for the conformation of the ribosome that precedes mRNA and tRNA movement on the small subunit, with a fully rotated 30S head, i.e., unliganded ribosome II (13). For the pretranslocation-like conformation of the ribosome, with a 30S subunit head not rotated, taken from a structure of the ribosome in complex with mRNA and a P-site tRNA mimic (47), the head domain of the 30S subunit, which adopts a different conformation compared to that in unliganded ribosome II, is shown in gray. The body of the 30S subunit in the pretranslocation-like state (not shown) is essentially identical in conformation to that in unliganded ribosome II (13, 47). The location of the mRNA downstream tunnel is indicated by the arrow.