Directed hydroxyl radical probing of 16S rRNA using Fe(II) tethered to ribosomal protein S4.

Abstract

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...

A Conserved Motif in Tetrahymena thermophila Telomerase Reverse Transcriptase Is Proximal to the RNA Template and Is Essential for Boundary Definition

“…In the present work, we employed site-directed Fe(II)-EDTA hydroxyl radical probing to map RNA-protein interactions in telomerase, as done previously for the ribosome (16,17). Our results demonstrated the CP2 motif and the TBE of Tetrahymena TER are in close proximity.…”

SAFA: Semi-automated footprinting analysis software for high-throughput quantification of nucleic acid footprinting experiments

“…Footprinting studies have provided a wealth of structural and functional information on nucleic acid behavior, and continue to be widely used in both qualitative and quantitative investigations. Among the numerous applications of footprinting approaches that probe secondary and tertiary structure are the mapping of DNA structure (Sun et al 1999;Guo and Tullius 2003), thermodynamic dissection of fundamental DNA-protein interactions (Ramesh and Nagaraja 1996;Gross et al 1998a,b;Brenowitz et al 2002), portraits of RNA folding pathways at millisecond time resolution (Sclavi et al 1997(Sclavi et al , 1998aDeras et al 2000;Brenowitz et al 2002;Uchida et al 2003), and high-resolution structural pictures of RNAprotein interactions in the ribosome (Heilek et al 1995;Wilson and Noller 1998;Joseph et al 2000).…”

Assembly of the 30S ribosomal subunit: Positioning ribosomal protein S13 in the S7 assembly branch

“…In prior experiments, S13 interactions with 16S rRNA were probed (Heilek and Noller 1996) and footprinted (Powers et al 1988a) in the presence of all the 1°b inding proteins (S4, S7, S8, S15, S17, and S20). Therefore, to understand which 1°binding protein(s) is required for S13 assembly, chemical modification and primer extension analysis were used to examine more minimal particles containing S13.…”