Directed hydroxyl radical probing of 16 S rRNA using Fe ( II ) tethered to ribosomal protein S 4 ( ribosomal RNA / chemical probing / ribosomes )

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. [FeCys31]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 419-432 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 ([Fe-Cys3t]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 ribonucleoprotein complex by using the Fenton reaction (12), and the positions of cleavage of the 16S rRNA chain are identified by primer extension (13). This approach provides certain advantages over previous methods for studying the RNA neighborhoods of ribosomal proteins. One shortcoming of chemical and enzymatic protection methods is that protection can be caused by proteininduced conformational rearrangements rather than direct protein-RNA contact. As a result, one cannot easily rule out the possibility that protected regions of the RNA might be remote from the site of protein-RNA contact. Crosslinking methods avoid this ambiguity but rely on favorable chemistry at the sites of crosslinking, limiting the scope of such approaches. Moreover, identification of crosslinked moieties often involve difficult and laborious procedures. Directed hydroxyl radical probing has the advantage that ribose moieties in virtually any region of the RNA backbone are susceptible to attack (14) and that the positions of cleavage can be located readily and assigned unambiguously to the proximity of the tethered Fe(II) ion. This general approach has been employed successfully to study DNA-protein interactions (1518). Our results show that Cys-31 of ribosomal protein S4 is in three-dimensional proximity to two discrete regions of the 16S rRNA chain, both ofwhich are located within its 5' domain and one of which has previously been localized by footprinting and crosslinking experiments to the S4 binding site itself. This approach should be generally useful for probing ribosome topography as well as that of other ribonucleoprotein complexes. MATERIALS AND METHODS Ribosomes, 30S ribosomal subunits, 16S rRNA, and ribosomal protein S4 were isolated as described (19, 20). Preparation of BABE was done as described (10). Formation of the FeBABE chelation complex was done by mixing 2 ,ul of freshly prepared 50mM FeSO4 with 18 ,ul of 11 mM BABE in 100mM NaOAc (pH 6.0), followed by incubation at room temperature for 30 min. Conjugation of S4 with Fe-BABE or with iodoacetate was carried out by mixing 15 ,ul of a 2.98 mg/ml solution of S4 (in 80 mM potassium Hepes, pH 7.7/1 M KCI/6 mM 2-mercaptoethanol) with 10 ,ul of 10 mM Fe-BABE or 10 ,l of 10 mM iodo[14C]acetate (Amersham; 5.93 mCi/mmol; 1 Ci = 37 GBq) in 1 M KCI/80mM Hepes, pH 7.7/0.01% Nikkol (total volume, 100 ,l), followed by incubation at 37°C for 15 min. Separation of the modified S4 from excess reagent was done by loading the reaction mixture on a 1-ml Sephadex G-50 spin-column (Pharmacia) preequilibrated with 1 M KCI/80 mM Hepes, pH 7.7/0.01% Nikkol and centrifuging for 4 min at 1700 x g on a clinical centrifuge. Derivatized S4 could be stored at -80°C for at least 4 weeks without loss of binding Abbreviation: BABE, 1-(p-bromoacetamidobenzyl)-EDTA. tPresent address: Department of Chemistry, Kenyon College, Gambier, OH 43022. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.