Kasugamycin (Ksg) specifically inhibits translation initiation of canonical but not of leaderless messenger RNAs. Ksg inhibition is thought to occur by direct competition with initiator transfer RNA. The 3.35-A structure of Ksg bound to the 30S ribosomal subunit presented here provides a structural description of two Ksg-binding sites as well as a basis for understanding Ksg resistance. Notably, neither binding position overlaps with P-site tRNA; instead, Ksg mimics codon nucleotides at the P and E sites by binding within the path of the mRNA. Coupled with biochemical experiments, our results suggest that Ksg indirectly inhibits P-site tRNA binding through perturbation of the mRNA-tRNA codon-anticodon interaction during 30S canonical initiation. In contrast, for 70S-type initiation on leaderless mRNA, the overlap between mRNA and Ksg is reduced and the binding of tRNA is further stabilized by the presence of the 50S subunit, minimizing Ksg efficacy.
Ribosome binding factor A (RbfA) is a bacterial cold shock response protein, required for an efficient processing of the 5' end of the 16S ribosomal RNA (rRNA) during assembly of the small (30S) ribosomal subunit. Here we present a crystal structure of Thermus thermophilus (Tth) RbfA and a three-dimensional cryo-electron microscopic (EM) map of the Tth 30S*RbfA complex. RbfA binds to the 30S subunit in a position overlapping the binding sites of the A and P site tRNAs, and RbfA's functionally important C terminus extends toward the 5' end of the 16S rRNA. In the presence of RbfA, a portion of the 16S rRNA encompassing helix 44, which is known to be directly involved in mRNA decoding and tRNA binding, is displaced. These results shed light on the role played by RbfA during maturation of the 30S subunit, and also indicate how RbfA provides cells with a translational advantage under conditions of cold shock.
In the initiation phase of bacterial translation, the 30S ribosomal subunit captures mRNA in preparation for binding with initiator tRNA. The purine-rich Shine-Dalgarno (SD) sequence, in the 5' untranslated region of the mRNA, anchors the 30S subunit near the start codon, via base pairing with an anti-SD (aSD) sequence at the 3' terminus of 16S rRNA. Here, we present the 3.3 A crystal structure of the Thermus thermophilus 30S subunit bound with an mRNA mimic. The duplex formed by the SD and aSD sequences is snugly docked in a "chamber" between the head and platform domains, demonstrating how the 30S subunit captures and stabilizes the otherwise labile SD helix. This location of the SD helix is suitable for the placement of the start codon AUG in the immediate vicinity of the mRNA channel, in agreement with reported crosslinks between the second position of the start codon and G1530 of 16S rRNA.
During 30S ribosomal subunit biogenesis, assembly factors are believed to prevent accumulation of misfolded intermediate states of low free energy that slowly convert into mature 30S subunits, namely, kinetically trapped particles. Among the assembly factors, the circularly permuted GTPase, RsgA, plays a crucial role in the maturation of the 30S decoding center. Here, directed hydroxyl radical probing and single particle cryo-EM are employed to elucidate RsgA΄s mechanism of action. Our results show that RsgA destabilizes the 30S structure, including late binding r-proteins, providing a structural basis for avoiding kinetically trapped assembly intermediates. Moreover, RsgA exploits its distinct GTPase pocket and specific interactions with the 30S to coordinate GTPase activation with the maturation state of the 30S subunit. This coordination validates the architecture of the decoding center and facilitates the timely release of RsgA to control the progression of 30S biogenesis.
dAlthough both tetracycline and tigecycline inhibit protein synthesis by sterically hindering the binding of tRNA to the ribosomal A site, tigecycline shows increased efficacy in both in vitro and in vivo activity assays and escapes the most common resistance mechanisms associated with the tetracycline class of antibiotics. These differences in activities are attributed to the tert-butylglycylamido side chain found in tigecycline. Our structural analysis by X-ray crystallography shows that tigecycline binds the bacterial 30S ribosomal subunit with its tail in an extended conformation and makes extensive interactions with the 16S rRNA nucleotide C1054. These interactions restrict the mobility of C1054 and contribute to the antimicrobial activity of tigecycline, including its resistance to the ribosomal protection proteins.T he ribosome, a central component of the protein synthesis machinery, is one of the major targets of clinically relevant antibiotics (1-3). In the last decade, crystal structures of a broad variety of antibiotics bound to either the large (50S) or the small (30S) subunit of the bacterial ribosome have been reported, unraveling their mechanism of action and demonstrating that they interact at a few distinct but functionally important sites (1, 2). For example, on the 50S subunit, antibiotics target primarily the peptidyl transferase center, the GTPase-associated center, or the ribosomal exit tunnel and hamper protein synthesis by interfering with the incorporation of new amino acids into the growing peptide chain (1). On the 30S subunit, antibiotics have thus far been observed at or near mRNA and tRNA binding sites and generally interfere with correct tRNA binding to the A site or with translocation of the tRNA/mRNA from the A site to the P site (1, 2). Tetracycline (TET) is an example of a 30S subunit binding antibiotic, with both structural and biochemical studies indicating that it binds the ribosome primarily in a pocket formed by the 16S rRNA helices 31 (h31) and 34, although secondary binding sites have also been observed (4-6). The significance of these secondary sites is unclear as binding to the primary site correlates best with the antimicrobial activity of the drug and resistance mutations (7).Upon their introduction into medicine in 1948, tetracyclines were quickly accepted because they offered a broad spectrum of activity (8). However, given the widespread use of "legacy" tetracyclines for more than 60 years, resistance in clinically important bacterial pathogens is common (8, 9). Accordingly, modern tetracycline derivatives, like tigecycline (TIG), omadacycline, and eravacycline (TP-434, Erv), have been developed and display activity against bacterial strains resistant to the legacy tetracyclines (3). TIG was the first representative of these derivatives to be approved for use by the FDA (10). Omadacycline is currently under clinical development for the treatment of acute bacterial skin infections (11), community-acquired bacterial pneumonia, and complicated urinary tract infection...
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