Effects of Tetracycline and Spectinomycin on the Tertiary Structure of Ribosomal RNA in the Escherichia coli 30 S Ribosomal Subunit

Abstract

Structural analysis of the 16 S rRNA in the 30 S subunit and 70 S ribosome in the presence of ribosome-specific antibiotics was performed to determine whether they produced rRNA structural changes that might provide further insight to their action. An UV cross-linking procedure that determines the pattern and frequency of intramolecular 16 S RNA cross-links was used to detect differences reflecting structural changes. Tetracycline and spectinomycin have specific effects detected by this assay. The presence of tetracycline inhibits the cross-link C967؋C1400 completely, increases the frequency of cross-link C1402؋1501 twofold, and decreases the cross-link G894؋U244 by one-half without affecting other cross-links. Spectinomycin reduces the frequency of the cross-link C934؋U1345 by 60% without affecting cross-linking at other sites. The structural changes occur at concentrations at which the antibiotics exert their inhibitory effects. For spectinomycin, the apparent binding site and the affected crosslinking site are distant in the secondary structure but are close in tertiary structure in several recent models, indicating a localized effect. For tetracycline, the apparent binding sites are significantly separated in both the secondary and the three-dimensional structures, suggesting a more regional effect.

16S rRNA Mutations That Confer Tetracycline Resistance inHelicobacter pyloriDecrease Drug Binding inEscherichia coliRibosomes

“…The nucleotide substitutions conferring resistance would not directly prohibit formation of these interactions but they may alter the conformation or flexibility of the backbone at positions 965 to 967 to weaken tetracycline binding. Rearrangements in the architecture of helix 31 during tetracycline binding were suggested by the fact that UV induced cross-linking between C1400 and C967 is inhibited completely by tetracycline (18). In terms of the resistance mutations, it is possible that pyrimidinerich sequences in helix 31 loop are not compatible with the tetracycline-induced conformation, leading to weakened tetracycline binding.…”

Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance

“…However, Schnappinger and Hillen (263) have pointed out that these apparent sites for drug interaction in the ribosome may not necessarily reflect the actual binding site. Indeed, interpretation of the probing studies referred to above is complicated by the observation that binding of tetracycline (which measures approximately 8 by 12 Å ) to the ribosome appears to cause wide-ranging structural change in 16 S rRNA (193). Furthermore, photoincorporation methods are subject to the limitation that upon irradiation, tetracycline photoproducts are generated which may react further with the ribosomes (196).…”

Mutations in the 16S rRNA Genes ofHelicobacter pyloriMediate Resistance to Tetracycline

“…Nucleotide 1054 contacts the A-site tRNA (40) and is very near G1058, the P. acnes tetracycline resistance mutation. Tetracycline binding also disrupts a 16S rRNA cross-link between C967 and C1400 in the E. coli 30S ribosomal subunit (21). The crystal structures of the 30S ribosome-tetracycline complexes show tetracycline interacting primarily with atoms of the phosphodiester backbone of nucleotides in h34 and the loop of h31, the boxed residues in Fig.…”