Mapping the rRNA neighborhood of the acceptor end of tRNA in the ribosome

Abstract

In order to map the rRNA environment of the acceptor end of tRNA in th e ribosome, hydroxyl radicals were generated in situ from Fe(II) attached via an EDTA linker to the 5' end of tRNA. Nucleotides in rRNA cleaved by the radicals were identified by primer extension, and assigned to the ribosomal A, P and E sites by standard criteria. In the A site, cleavages were found in the 2555-2573 region of 23S rRNA, around bases previously shown to be protected by A site tRNA, and in the alpha-sarcin loop, the site of interaction of elongation factors EF-Tu and EF-G. P site cleavages occurred in the 2250 loop, where a base pair is made with C74 of tRNA; and around the 2493 region in domain V. Interestingly, two clusters of nucleotides in 23S rRNA are accessible to both A site and P site tRNA probes. The first cluster is in the 1940-1965 region of domain IV, around the site of affinity labeling by the 3' end of tRNA, and the second cluster is around the bulged adenosine A2602, whose accessibility to chemical probes is enhanced by P site tRNA and decreased by A site tRNA. From the E site, cleavages occur in the 2390-2440 region, surrounding C2394, a base protected from dimethyl sulfate by E site tRNA, and in the phylogenetically variable stem at positions 1860/1880 of domain IV. Unexpectedly, no cleavages were detected in the central loop of domain V of 23S rRNA.