Contacts between the growing peptide chain and the 23S RNA in the 50S ribosomal subunit

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Abstract
Peptides of defined length carrying a diazirine photo-affinity label attached either to the α-NH2 group of the N-terminal methionine residue, or to the £-NH2 group of an immediately adjacent lysine residue, were prepared in situ on Escherichia coli ribosomes in the presence of a synthetic mRNA analogue. Peptide growth was stopped simply by withholding the aminoacyl-tRNA cognate to an appropriate downstream codon. After photo-activation at 350 nm the sites of cross-linking to ribosomal RNA were determined by our standard procedures; the C-terminal amino acid of each peptide was labelled with tritium, in order to confirm whether the individual cross-linked complexes contained the expected ‘full-length’ peptide, as opposed to shorter products. The shortest peptides became cross-linked to sites within the ‘peptidyl transferase ring’ of the 23S RNA, namely to positions 2062, 2506, 2585 and 2609. However, already when the peptide was three or four residues long, a new crosslink was observed several hundred nucleotides away in another secondary structural domain; this site, at position 1781, lies within one of several RNA regions which have been implicated in other studies as being located close to the peptidyl transferase ring. Further application of this approach, combined with model-building studies, should enable the path of the nascent peptide through the large ribosomal subunit to be definitively mapped.