HflX is a GTP binding protein of unknown function. Based on the presence of the hflX gene in hflA operon, HflX was believed to be involved in the lytic-lysogenic decision during phage infection in Escherichia coli. We find that E. coli HflX binds 16S and 23S rRNA – the RNA components of 30S and 50S ribosomal subunits. Here, using purified ribosomal subunits, we show that HflX specifically interacts with the 50S. This finding is in line with the homology of HflX to GTPases involved in ribosome biogenesis. However, HflX-50S interaction is not limited to a specific nucleotide-bound state of the protein, and the presence of any of the nucleotides GTP/GDP/ATP/ADP is sufficient. In this respect, HflX is different from other GTPases. While E. coli HflX binds and hydrolyses both ATP and GTP, only the GTP hydrolysis activity is stimulated by 50S binding. This work uncovers interesting attributes of HflX in ribosome binding.

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.

EngA, a unique GTPase containing a KH-domain preceded by two consecutive G-domains, displays distinct nucleotide binding and hydrolysis activities. So far, Escherichia coli EngA is reported to bind the 50S ribosomal subunit in the guanosine-5′-trihosphate (GTP) bound state. Here, for the first time, using mutations that allow isolating the activities of the two G-domains, GD1 and GD2, we show that apart from 50S, EngA also binds the 30S and 70S subunits. We identify that the key requirement for any EngA–ribosome association is GTP binding to GD2. In this state, EngA displays a weak 50S association, which is further stabilized when GD1 too binds GTP. Exchanging bound GTP with guanosine-5′-diphosphate (GDP), at GD1, results in interactions with 50S, 30S and 70S. Therefore, it appears that GD1 employs GTP hydrolysis as a means to regulate the differential specificity of EngA to either 50S alone or to 50S, 30S and 70S subunits. Furthermore, using constructs lacking either GD1 or both GD1 and GD2, we infer that GD1, when bound to GTP and GDP, adopts distinct conformations to mask or unmask the 30S binding site on EngA. Our results suggest a model where distinct nucleotide-bound states of the two G-domains regulate formation of specific EngA–ribosome complexes.

The impact of Nuclear Magnetic Resonance (NMR) on studies of large macromolecular complexes hinges on improvements in sensitivity and resolution. Dynamic nuclear polarization (DNP) in the solid state can offer improved sensitivity, provided sample preparation is optimized to preserve spectral resolution. For a few nanomoles of intact ribosomes and an 800 kDa ribosomal complex we demonstrate that the combination of DNP and magic-angle spinning NMR (MAS-NMR) allows one to overcome current sensitivity limitations so that homo-and heteronuclear 13 C and 15 N NMR correlation spectra can be recorded. Ribosome particles, directly pelleted and frozen into an NMR rotor, yield DNP signal enhancements on the order of *25-fold and spectra that exhibit narrow linewidths, suitable for obtaining site-specific information. We anticipate that the same approach is applicable to other high molecular weight complexes.

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...