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.Spectinomycin, a broad-spectrum antibiotic that selectively targets bacterial ribosomes (1-3), remains important in clinical and veterinary use, as it induces only low levels of bacterial resistance (4-6). Despite decades of worldwide use of the drug, the molecular mechanism by which spectinomycin inhibits translation remains unclear.Spectinomycin, an aminocyclitol constituted from two glucose moieties (7) (Figure 1, panel a), inhibits translocation of transfer RNAs (tRNAs) and messenger RNA (mRNA) on the ribosome (8-10), the step that follows the formation of each peptide bond. Translocation, catalyzed by the GTPase elongation factor G (EF-G), involves multiple large conformational * Corresponding author, jcate@lbl.gov.. Supporting Information Available:This material is available free of charge via the Internet.Accession Codes: The coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 2QOU, 2QOV, 2QOW, and 2QOX (70S ribosome in complex with spectinomycin), and 2QOY, 2QOZ, 2QP0, and 2QP1 (70S ribosome in complex with spectinomycin and neomycin). HHS Public Access Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript changes in the ribosome (9,(11)(12)(13)(14)(15). Translocation is thought to begin with a ratchet-like motion of the small (30S) ribosomal subunit relative to the large (50S) ribosomal subunit (11,15,16), followed by swiveling of the head of the small subunit (12, 13) and "unlocking" (opening) of the tRNA binding groove to allow peptidyl (P-site) tRNA to pass into the Exit (E) site (13,14,17,18). These conformational changes lead to discrete movements of bound tRNAs, where the tRNAs move first with respect to the 50S subunit into hybrid binding states and then move with respect to the 30S subunit (19). Unlike most of the translocation inhibitors, spectinomycin slows but does not completely abolish multiple-turnover translocation (9). It also does not induce mRNA miscoding (20, 21).The binding site for spectinomycin, in RNA helix 34 (h34) of the head domain of the 30S subunit, resides near the single ribosomal RNA (rRNA) helix that connects the head domain to the rest of the small subunit (i.e., neck of the 30S subunit) and faces the mRNA binding groove (22-26) (Figure 1, panels b and c; Supplementary Figure 1). Biochemical data (9,24,25,27) and the structure of spectinomycin bound to the 30S ribosomal subunit (26) suggested ...

Molecular mechanics of 30S subunit head rotation

“…Next, measuring the deviation of positions along the helical axis describing the core of the head, we localized the origins of head movement to two hinge points, located at the middle of h28 and the end of h35. These results show that h28, which has long been implicated in head rotation (11,25,(36)(37)(38)(39), is indeed involved in head movement, although not as the center of rotation.…”

“…Assignment of the origins of head rotation to these two hinge points was further validated by the strong correlation between the magnitudes of their deflection angles and the degree of head rotation in the different structures. Finally, our findings explain the inhibition of translocation by the antibiotic spectinomycin, which binds to the pivot point of hinge 2 (24,25,29,30). …”

“…Spectinomycin binds within the 30S head domain (29), trapping the head in a partially swiveled state (24,25) and drastically reducing the rate of translocation (35). Chemical footprinting (29), mutational analysis (29,30,45), and crystal structures of ribosomes (25) or 30S subunits (24) in complex with spectinomycin localized its binding site to G1064, C1066, G1068, A1191, C1192, and G1193 at the h34/h35 junction. Our findings show that these contact positions with spectinomycin are centered on the articulation point of hinge 2 ( Fig.…”

“…These findings provide evidence that small-subunit head rotation plays a central role in the second step of translocation. A full description of the molecular mechanism of translocation will therefore depend on an understanding of the structural basis of head rotation.As has been widely believed (11,17,(23)(24)(25)(26), we anticipated that the axis of head rotation would pass through helix h28 of 16S rRNA (often called the "neck"), the sole covalent connection between the head and body of the 30S subunit. Using the Euler-Rodrigues (E-R) method (27, 28), we localized the axis of head rotation for structures of 55 ribosome complexes, covering a range of more than 20°of head rotation.…”

Antiviral Activity of Geneticin against Bovine Viral Diarrhoea Virus

“…This is somewhat different from the ability of those aminoglycosides that inhibit protein synthesis by binding to the A site of 30S ribosomes [22], where the ability of G418 binding to RNA is assigned to its ring II [23]. Moreover, although it was recently demonstrated that G418, gentamicin and other related aminoglycosides could suppress stop codons and induce frame-shifting [24][25][26][27], only G418 was shown to inhibit virus-induced cytopathology, suggesting that the antiviral activity of G418 is different from inhibition or deregulation of translation.…”