Tetracycline analogs fell into two classes on the basis of their mode of action. Tetracycline, chlortetracycline, minocycline, doxycycline, and 6-demethyl-6-deoxytetracycline inhibited cell-free translation directed by either Escherichia coli or Bacillus subtilis extracts. A second class of analogs tested, including chelocardin, anhydrotetracycline, 6-thiatetracycline, anhydrochlortetracycline, and 4-epi-anhydrochlortetracycline, failed to inhibit protein synthesis in vitro or were very poor inhibitors. Tetracyclines of the second class, however, rapidly inhibited the in vivo incorporation of precursors into DNA and RNA as well as protein. The class 2 compounds therefore have a mode of action that is entirely distinct from the class 1 compounds, such as tetracycline that are used clinically. Although tetracyclines of the second class entered the cytoplasm, the ability of these analogs to inhibit macromolecular synthesis suggests that the cytoplasmic membrane is their primary site of action. The interaction of class 1 and class 2 tetracyclines with ribosomes was studied by examining their effects on the chemical reactivity of bases in 16S rRNA to dimethyl sulfate. Class 1 analogs affected the reactivity of bases to dimethyl sulfate. The response with class 2 tetracyclines varied, with some analogs affecting reactivity and others (chelocardin and 4-epi-anhydrotetracycline) not.The tetracyclines are a group of broad-spectrum antibiotics which are generally considered to prevent bacterial growth by inhibiting protein synthesis. This results from binding of antibiotic to a single site in the 30S ribosomal subunit which prevents attachment of aminoacyl tRNA to the ribosomal acceptor site (3). In order to reach the ribosome, these antibiotics must traverse the hydrophobic lipid bilayer of the bacterial cytoplasmic membrane (3). At physiological pH tetracyclines can exist as an equilibrium mixture of two free base forms: a low-energy, lipophilic nonionized species and a high-energy, hydrophilic zwitterionic structure (7). A solvent-dependent equilibrium between the two forms has been demonstrated with oxytetracycline free base and is supported by X-ray analyses of tetracyclines crystallized from aqueous and nonaqueous solvents (7,15). Both forms are believed to be important for the antibacterial activity of tetracyclines, the low-energy, lipophilic conformational form (Fig. 1A) for uptake across the cytoplasmic membrane and the hydrophilic, zwitterionic structure (Fig. 1B) for binding to the ribosome (7).Chelocardin (14) is a naturally occurring anhydrotetracycline derivative with a modified A ring (Fig. 2). In contrast to the solvent-dependent equilibrium of the two tetracycline species mentioned above, chelocardin apparently exists in the same conformation in both polar and nonpolar solvents, as evidenced by circular dichroism measurements (6). We believe this is related to the planarity of the BCD rings in chelocardin and that a lipophilic form, perhaps related to that of tetracycline, is the preferred species. Thes...

Aminoglycoside inhibitors of translation have been shown previously to inhibit in vitro self-splicing by group I introns. Chemical probing of the phage T4-derived sunY intron shows that neomycin, streptomycin, and related antibiotics protected the N-7 position of G96, a universally conserved guanine in the binding site for the guanosine cofactor in the splicing reaction. The antibiotics also disrupted structural contacts that have been proposed to bring the 5' cleavage site of the intron into proximity to the catalytic core. In contrast, the strictly competitive inhibitors deoxyguanosine and arginine protected only the N-7 position of G96. Parallels between these results and previously observed protection of 16S ribosomal RNA by aminoglycosides raise the possibility that group I intron splicing and transfer RNA selection by ribosomes involve similar RNA structural motifs.

Previous studies suggest that the mechanism of action of the ribosome in translation involves crucial transfer RNA (tRNA)-ribosomal RNA (rRNA) interactions. Here, a selection scheme was developed to identify bases in 16S rRNA that are essential for tRNA binding to the P site of the small (30S) ribosomal subunit. Modification of the N-1 and N-2 positions of 2-methylguanine 966 and of the N-7 position of guanine 1401 interfered with messenger RNA (mRNA)-dependent binding of tRNA to the P site. Modification of the same positions as well as of the N-1 and N-2 positions of guanine 926 interfered with mRNA-independent binding of tRNA at high magnesium ion concentration. These results suggest that these three bases are involved in intermolecular contacts between ribosomes and tRNA.

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