Structural basis for aminoglycoside inhibition of bacterial ribosome recycling.

Aminoglycosides are widely used antibiotics that cause messenger RNA decoding errors, block mRNA and transfer RNA translocation, and inhibit ribosome recycling. Ribosome recycling follows the termination of protein synthesis and is aided by ribosome recycling factor (RRF) in bacteria. The molecular mechanism by which aminoglycosides inhibit ribosome recycling is unknown. Here we show in X-ray crystal structures of the Escherichia coli 70S ribosome that RRF binding causes RNA helix H69 of the large ribosomal subunit, which is crucial for subunit association, to swing away from the subunit interface. Aminoglycosides bind to H69 and completely restore the contacts between ribosomal subunits that are disrupted by RRF. These results provide a structural explanation for aminoglycoside inhibition of ribosome recycling.

Structural analysis of kasugamycin inhibition of translation.

The prokaryotic ribosome is an important target of antibiotic action. We determined the X-ray structure of the aminoglycoside kasugamycin (Ksg) in complex with the Escherichia coli 70S ribosome at 3.5-A resolution. The structure reveals that the drug binds within the messenger RNA channel of the 30S subunit between the universally conserved G926 and A794 nucleotides in 16S ribosomal RNA, which are sites of Ksg resistance. To our surprise, Ksg resistance mutations do not inhibit binding of the drug to the ribosome. The present structural and biochemical results indicate that inhibition by Ksg and Ksg resistance are closely linked to the structure of the mRNA at the junction of the peptidyl-tRNA and exit-tRNA sites (P and E sites).

Chemically related 4,5-linked aminoglycoside antibiotics drive subunit rotation in opposite directions.

Dynamic remodelling of intersubunit bridge B2, a conserved RNA domain of the bacterial ribosome connecting helices 44 (h44) and 69 (H69) of the small and large subunit, respectively, impacts translation by controlling intersubunit rotation. Here we show that aminoglycosides chemically related to neomycin-paromomycin, ribostamycin and neamine-each bind to sites within h44 and H69 to perturb bridge B2 and affect subunit rotation. Neomycin and paromomycin, which only differ by their ring-I 6'-polar group, drive subunit rotation in opposite directions. This suggests that their distinct actions hinge on the 6'-substituent and the drug's net positive charge. By solving the crystal structure of the paromomycin-ribosome complex, we observe specific contacts between the apical tip of H69 and the 6'-hydroxyl on paromomycin from within the drug's canonical h44-binding site. These results indicate that aminoglycoside actions must be framed in the context of bridge B2 and their regulation of subunit rotation.

Allosteric control of the ribosome by small-molecule antibiotics.

Protein synthesis is targeted by numerous, chemically distinct antibiotics that bind and inhibit key functional centers of the ribosome. Using single-molecule imaging and X-ray crystallography, we show that the aminoglycoside neomycin blocks aminoacyl-transfer RNA (aa-tRNA) selection and translocation as well as ribosome recycling by binding to helix 69 (H69) of 23S ribosomal RNA within the large subunit of the Escherichia coli ribosome. There, neomycin prevents the remodeling of intersubunit bridges that normally accompanies the process of subunit rotation to stabilize a partially rotated ribosome configuration in which peptidyl (P)-site tRNA is constrained in a previously unidentified hybrid position. Direct measurements show that this neomycin-stabilized intermediate is incompatible with the translation factor binding that is required for distinct protein synthesis reactions. These findings reveal the functional importance of reversible intersubunit rotation to the translation mechanism and shed new light on the allosteric control of ribosome functions by small-molecule antibiotics.

Structures of the bacterial ribosome in classical and hybrid states of tRNA binding.

During protein synthesis, the ribosome controls the movement of tRNA and mRNA by means of large-scale structural rearrangements. We describe structures of the intact bacterial ribosome from Escherichia coli that reveal how the ribosome binds tRNA in two functionally distinct states, determined to a resolution of ~3.2 angstroms by means of x-ray crystallography. One state positions tRNA in the peptidyl-tRNA binding site. The second, a fully rotated state, is stabilized by ribosome recycling factor and binds tRNA in a highly bent conformation in a hybrid peptidyl/exit site. The structures help to explain how the ratchet-like motion of the two ribosomal subunits contributes to the mechanisms of translocation, termination, and ribosome recycling.

Structural basis for mRNA and tRNA positioning on the ribosome.

Protein synthesis requires the accurate positioning of mRNA and tRNA in the peptidyl-tRNA site of the ribosome. Here we describe x-ray crystal structures of the intact bacterial ribosome from Escherichia coli in a complex with mRNA and the anticodon stem-loop of P-site tRNA. At 3.5-A resolution, these structures reveal rearrangements in the intact ribosome that clamp P-site tRNA and mRNA on the small ribosomal subunit. Binding of the anticodon stem-loop of P-site tRNA to the ribosome is sufficient to lock the head of the small ribosomal subunit in a single conformation, thereby preventing movement of mRNA and tRNA before mRNA decoding.

Mixing crowded biological solutions in milliseconds.

In vitro studies of biological reactions are rarely performed in conditions that reflect their native intracellular environments where macromolecular crowding can drastically change reaction rates. Kinetics experiments require reactants to be mixed on a time scale faster than that of the reaction. Unfortunately, highly concentrated solutions of crowding agents such as bovine serum albumin and hemoglobin that are viscous and sticky are extremely difficult to mix rapidly. We demonstrate a new droplet-based microfluidic mixer that induces chaotic mixing of crowded solutions in milliseconds due to protrusions of the microchannel walls that generate oscillating interfacial shear within the droplets. Mixing in the microfluidic mixer is characterized, mechanisms underlying mixing are discussed, and evidence of biocompatibility is presented. This microfluidic platform will allow for the first kinetic studies of biological reactions with millisecond time resolution under conditions of macromolecular crowding similar to those within cells.

Structures of the bacterial ribosome at 3.5 A resolution.

We describe two structures of the intact bacterial ribosome from Escherichia coli determined to a resolution of 3.5 angstroms by x-ray crystallography. These structures provide a detailed view of the interface between the small and large ribosomal subunits and the conformation of the peptidyl transferase center in the context of the intact ribosome. Differences between the two ribosomes reveal a high degree of flexibility between the head and the rest of the small subunit. Swiveling of the head of the small subunit observed in the present structures, coupled to the ratchet-like motion of the two subunits observed previously, suggests a mechanism for the final movements of messenger RNA (mRNA) and transfer RNAs (tRNAs) during translocation.

X-ray crystal structures of the WT and a hyper-accurate ribosome from Escherichia coli.

Protein biosynthesis on the ribosome requires accurate reading of the genetic code in mRNA. Two conformational rearrangements in the small ribosomal subunit, a closing of the head and body around the incoming tRNA and an RNA helical switch near the mRNA decoding site, have been proposed to select for complementary base-pairing between mRNA codons and tRNA anticodons. We determined x-ray crystal structures of the WT and a hyper-accurate variant of the Escherichia coli ribosome at resolutions of 10 and 9 A, respectively, revealing that formation of the intact 70S ribosome from its two subunits closes the conformation of the head of the small subunit independent of mRNA decoding. Moreover, no change in the conformation of the switch helix is observed in two steps of tRNA discrimination. These 70S ribosome structures indicate that mRNA decoding is coupled primarily to movement of the small subunit body, consistent with previous proposals, whereas closing of the head and the helical switch may function in other steps of protein synthesis.