Photo-affinity labelling at the peptidyl transferase centre reveals two different positions for the A- and P-sites in domain V of 23S rRNA.

Abstract

Photo‐reactive 3‐(4′‐benzoylphenyl)propionyl‐Phe‐tRNA bound to the A‐ or the P‐site was crosslinked to 23S RNA upon irradiation at 320 nm. The sites of reaction were identified as U‐2584 and U‐2585 at the A‐site and A‐2451 and C‐2452 at the P‐site. Minor crosslinks from both sites were observed at nucleotides A‐2503 to U‐2506. All sites identified lie in close proximity according to the secondary structure model and constitute part of the highly conserved loop region of domain V. Antibiotics known to inhibit peptidyl transferase activity had a pronounced effect on photo‐crosslinking. In addition, tetracycline was also shown to photo‐crosslink to this region. These experiments permit a dissection of the peptidyl transferase region on the 23S RNA into two distinct areas for the A‐ and P‐site.

The three-dimensional folding of ribosomal RNA; localization of a series of intra-RNA cross-links in 23S RNA induced by treatment ofEscherichia coli50S ribosomal subunits withbis-(2-chloroethyl)-methylamine

“…The first concerns the 'ring' structures, such as the peptidyl transferase ring (15,16) formed between helices 73, 74, 89, 90 and 93 (marked 'PT' in Figure 4). Similar structures occur in many places throughout the 16S and 23S RNA, and involve relatively high numbers of single-stranded residues.…”

“…Similar structures occur in many places throughout the 16S and 23S RNA, and involve relatively high numbers of single-stranded residues. The peptidyl transferase ring for example includes no less than thirty single-stranded bases of adjacent oligonucleotides in both the ribonuclease A and T, (Figure 4), but the functional data (14)(15)(16) indicate a similar degree of compactness in the organisation of neighbouring single-stranded loop structures, N1I being particularly noteworthy because it forms a direct link between bases that have been identified by foot-printing (14) as part of the ribosomal E-site for tRNA.…”

“…The 3'-terminal A-residue of tRNA has been cross-linked to G-1945 (in helix 71) and to protein L27 (24), which in turn has cross-links to the 23S RNA in helices 81 and 83-84 (6). The region of helices 65-71 is connected to the peptidyl transferase region by the cross-links U0, U12 and U14, and the peptidyl transferase ring itself is the target of reaction with affinity analogues of the amino acid in aminoacyl tRNA (16). Cross-link U5 (Figure 4) brings helix 35 into direct juxtaposition with this functional area.…”

“…The data obtained include nine 'secondary' and seven 'tertiary' cross-links. The latter impose significant new constraints on the tertiary folding of the 23S RNA, particularly in the conserved secondary structural 'core' (12, 13) of the molecule, and define a number of new RNA neighbourhoods in those regions of the 50S subunit which have been associated with tRNA binding and peptidyl transferase activity (14)(15)(16).…”

Incorporation of Dinitrophenyl Protein L23 into Totally Reconstituted Escherichia coli 50 S Ribosomal Subunits and Its Localization at Two Sites by Immune Electron Microscopy

“…Puromycin-modified L23 has been incorporated into reconstituted 50 S subunits, with a proportional decrease in mRNA-dependent aminoacyl-tRNA binding activity but no specific effect on the peptidyltransferase reaction (6). Tetracycline interacts directly with the central loop of domain V, the region of 23 S rRNA most strongly implicated in peptidyltransferase activity (43,44), and it also specifically stimulates incorporation of puromycin into protein L23 (7). Streptomycin affects the elongation step of protein synthesis primarily by interfering in ternary complex binding.…”