Cysteine residues were introduced into three different positions distributed on the surface of ribosomal protein S5, to serve as targets for derivatization with an Fe(II)-ethyl-enediaminetetraacetic acid linker. Hydroxyl radicals generated locally from the tethered Fe(II) in intermediate ribonucleoprotein particles or in 30S ribosomal subunits reconstituted from derivatized S5 caused cleavage of the RNA, resulting in characteristically different cleavage patterns for the three different tethering positions. These findings provide constraints for the three-dimensional folding of 16S ribosomal RNA (rRNA) and for the orientation of S5 in the 30S subunit, and they further suggest that antibiotic resistance and accuracy mutations in S5 may involve perturbation of 16S rRNA.

A refined model has been developed for the folding of 16S rRNA in the 30S subunit, based on additional constraints obtained from new experimental approaches. One set of constraints comes from hydroxyl radical footprinting of each of the individual 30S ribosomal proteins, using free Fe(2+)-EDTA complex. A second approach uses localized hydroxyl radical cleavage from a single Fe2+ tethered to unique positions on the surface of single proteins in the 30S subunit. This has been carried out for one position on the surface of protein S4, two on S17, and three on S5. Nucleotides in 16S rRNA that are essential for P-site tRNA binding were identified by a modification interference strategy. Ribosomal subunits were partially inactivated by chemical modification at a low level. Active, partially modified subunits were separated from inactive ones by binding 3'-biotinderivatized tRNA to the 30S subunits and captured with streptavidin beads. Essential bases are those that are unmodified in the active population but modified in the total population. The four essential bases, G926, 2mG966, G1338, and G1401 are a subset of those that are protected from modification by P-site tRNA. They are all located in the cleft of our 30S subunit model. The rRNA neighborhood of the acceptor end of tRNA was probed by hydroxyl radical probing from Fe2+ tethered to the 5' end of tRNA via an EDTA linker. Cleavage was detected in domains IV, V, and VI of 23S rRNA, but not in 5S or 16S rRNA. The sites were all found to be near bases that were protected from modification by the CCA end of tRNA in earlier experiments, except for a set of E-site cleavages in domain IV and a set of A-site cleavages in the alpha-sarcin loop of domain VI. In vitro genetics was used to demonstrate a base-pairing interaction between tRNA and 23S rRNA. Mutations were introduced at positions C74 and C75 of tRNA and positions 2252 and 2253 of 23S rRNA. Interaction of the CCA end of tRNA with mutant ribosomes was tested using chemical probing in conjunction with allele-specific primer extension. The interaction occurred only when there was a Watson-Crick pairing relationship between positions 74 of tRNA and 2252 of 23S rRNA. Using a novel chimeric in vitro reconstitution method, it was shown that the peptidyl transferase reaction depends on this same Watson-Crick base pair.

Localized hydroxyl radical probing has been used to explore the rRNA neighborhood around a unique position in the structure of the Escherichia coli 30S ribosomal subunit. Fe(II) was attached to ribosomal protein S4 at Cys-31 via the reagent 1-(p-bromoacetamidobenzyl)-EDTA. [Fe-Cys31] S4 was then complexed with 16S rRNA or incorporated into active 30S ribosomal subunits by in vitro reconstitution with 16S rRNA and a mixture of the remaining 30S subunit proteins. Hydroxyl radicals generated from the tethered Fe resulted in cleavage of the 16S rRNA chain in two localized regions of its 5' domain. One region spans positions [419][420][421][422][423][424][425][426][427][428][429][430][431][432] and is close to the multihelix junction previously placed at the RNA binding site of S4 by chemical and enzymatic protection (footprinting) and crosslinking studies. A second site of directed cleavage includes nucleotides 297-303, which overlap a site that is protected from chemical modification by protein S16, a near neighbor of S4 in the ribosome. These results provide useful information about the three-dimensional organization of 16S rRNA and indicate that these two regions of its 5' domain are in close spatial proximity to Cys-31 of protein S4.Understanding the molecular mechanism of translation depends on detailed knowledge of the three-dimensional structure of the ribosome. In the absence of an x-ray crystal structure, a wide variety of alternative biochemical and physical approaches have been devised to obtain information concerning the relative locations of ribosomal proteins and rRNA and other macromolecular components of translation in the ribosome (1). Indeed, even with a well-resolved electron density map in hand, information of this kind will likely be essential for its interpretation.In the studies presented here, we describe a biochemical method for obtaining information about the three-dimensional structure of RNA-protein complexes such as the ribosome. It consists of generating hydroxyl radicals locally from Fe(II) tethered to a single position in the ribosome, which results in cleavage of the rRNA backbone at positions that are in close proximity to the Fe(II) ion. Because of the short lifetime of hydroxyl radicals in aqueous solution, cleavage is usually restricted to positions in the RNA that are within about 10 A of the Fe(II) ion (2, 3). We use ribosomal protein S4 as a model system for these studies. The binding of S4 to 16S rRNA has been extensively characterized (4-8); it is one of six small-subunit ribosomal proteins that bind specifically to the RNA in the absence of the other proteins (9). Fe(II) is tethered to the unique cysteine residue at position 31 of protein S4 via 1-(p-bromoacetamidobenzyl)-EDTA (BABE), a reagent that has successfully been used to map intramolecular proximities in proteins (10, 11). Iron-derivatized protein S4 S4) is then bound to 16S rRNA, either alone, or in a fully assembled 30S ribosomal subunit. Hydroxyl radicals are generated after assembly of the ribonucleopro...

The binding site and probable site of action have been determined for the universal antibiotic amicetin which inhibits peptide bond formation. Evidence from in vivo mutants, site‐directed mutations and chemical footprinting all implicate a highly conserved motif in the secondary structure of the 23S‐like rRNA close to the central circle of domain V. We infer that this motif lies at, or close to, the catalytic site in the peptidyl transfer centre. The binding site of amicetin is the first of a group of functionally related hexose‐cytosine inhibitors to be localized on the ribosome.

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