Transit of tRNA through the Escherichia coli ribosome - Cross-linking of the 3 ' end of tRNA to specific nucleotides of the 23 S ribosomal RNA at theA, P, and E sites

When bound to Escherichia coli ribosomes and irradiated with near-UV light, various derivatives of yeast tRNA(Phe) containing 2-azidoadenosine at the 3' terminus form cross-links to 23 S rRNA and 50 S subunit proteins in a si te-dependent manner. A and P site-bound tRNAs, whose 3' termini reside in t he peptidyl transferase center, label primarily nucleotides U2506 and U2585 and protein L27. In contrast, E site-bound tRNA labels nucleotide C2422 an d protein L33. The cross-linking patterns confirm the topographical separat ion of the peptidyl transferase center from the E site domain. The relative amounts of label incorporated into the universally conserved residues U250 6 and U2585 depend on the occupancy of the A and P sites by different tRNA ligands and indicates that these nucleotides play a pivotal role in peptide transfer. In particular, the S'-adenosine of the peptidyl-tRNA analogue, A cPhe-tRNA(Phe), remains,in close contact with U2506 regardless of whether i ts anticodon is located in the A site or P site. Our findings, therefore, m odify and extend the hybrid state model of tRNA-ribosome interaction. We sh ow that the S'-end of the deacylated tRNA that is formed after transpeptida tion does not immediately progress to the E site but remains temporarily in the peptidyl transferase center. In addition, we demonstrate that the E si te, defined by the labeling of nucleotide C2422 and protein L33, represents an intermediate state of binding that precedes the entry of deacylated tRN A into the F (final) site from which it dissociates into the cytoplasm.