Abstract
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 site-dependent manner. A and P site-bound tRNAs, whose 3' termini reside in the peptidyl transferase center, label primarily nucleotides U2506 and U2585 and protein L27. In contrast, E site-bound tRNA labels nucleotide C2422 and protein L33. The cross-linking patterns confirm the topographical separation of the peptidyl transferase center from the E site domain. The relative amounts of label incorporated into the universally conserved residues U2506 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 3'-adenosine of the peptidyl-tRNA analogue, AcPhe-tRNA(Phe), remains in close contact with U2506 regardless of whether its anticodon is located in the A site or P site. Our findings, therefore, modify and extend the hybrid state model of tRNA-ribosome interaction. We show that the 3'-end of the deacylated tRNA that is formed after transpeptidation does not immediately progress to the E site but remains temporarily in the peptidyl transferase center. In addition, we demonstrate that the E site, defined by the labeling of nucleotide C2422 and protein L33, represents an intermediate state of binding that precedes the entry of deacylated tRNA into the F (final) site from which it dissociates into the cytoplasm.
Highlights
When bound to Escherichia coli ribosomes and irradiated with near-UV light, various derivatives of yeast tRNAPhe containing 2-azidoadenosine at the 3 terminus form cross-links to 23 S rRNA and 50 S subunit proteins in a site-dependent manner
Defining the Nucleotides of the 23 S rRNA At the Peptidyl Transferase Center—Incorporation of 2-azidoadenosine into tRNA yields a photoreactive derivative, (2N3A76)tRNAPhe, which can be aminoacylated, will bind to ribosomes, is able to participate in peptide bond formation and, when irradiated with near-UV light, can form 2- to 3-Å cross-links to other macromolecules in its immediate vicinity [3, 14]
In our earlier studies, (2N3A76)tRNAPhe was primarily used for the identification of ribosomal proteins that serve as topographical markers of the A, P, and E sites on the E. coli ribosome
Summary
Materials—The sources of tRNAs, enzymes, and radioactively labeled compounds were as described previously [4, 12, 13]. B Phe-tRNA bound to ribosomal A/T or R site as ternary complex with EF-Tu and GMPPNP. Formation of tRNA1⁄7Ribosome Complexes—Binding of [5Ј-32P] (2N3A76)tRNAPhe derivatives to the ribosomal P, A, R, and E sites was carried out according to established procedures [2, 3, 17, 19] and measured by filter retention [20]. 9 –13% of the non-covalently bound (2N3A76)tRNAPhe became covalently cross-linked to the ribosomes under our conditions [3, 4, 13]. Isolation and Analysis of Cross-linked Complexes—Sites in the 23 S rRNA labeled by (2N3A76)tRNAPhe were identified by treatment with RNase H in the presence of selected pairs of oligodeoxyribonucleotides [23], RNase T1 protection experiments [24], and primer extension analysis [25, 26]. The oligodeoxyribonucleotides used in these assays were complementary to sequences within the 23 S rRNA encompassing nucleotides 2776 –2790 (oligo 1), 2616 –2630 (oligo 2), 2582–2597 (oligo 2a), 2563–2577 (oligo 3), 2532–2546 (oligo 3a), 2492–2506 (oligo 3b), 2438 – 2452 (oligo 4), 2354 –2368 (oligo 4a), 2229 –2243 (oligo 5), 2110 –2124 (oligo 5a), 2084 –2098 (oligo 6), 2050 –2064 (oligo 6a), 2018 –2032 (oligo 7), 1983–1997 (oligo 7a), 1903–1917 (oligo 8), 1806 –1820 (oligo 8a), 1703–1717 (oligo 9), 2570 –2591 (oligo 10), 2489 –2522 (oligo 11), 2497– 2512 (oligo 12), 2448 –2468 (oligo 13), 2416 –2443 (oligo 14), 2402–2434 (oligo 15), 2385–2408 (oligo 16), 2362–2381 (oligo 17), 2058 –2080 (oligo 18), 1951–1980 (oligo 19), 1931–1957 (oligo 20), and 1923–1947 (oligo 21). 50 S-subunit proteins cross-linked to (2N3A76)tRNAPhe were ana-
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