Photoaffinity polyamines:: interactions with AcPhe-tRNA free in solution or bound at the P-site of Escherichia coli ribosomes

Two photoreactive derivatives of spermine, azido-benzamidino (ABA)-spermine and atidonitrobenzoyl (ANB)-spermine, were used for mapping of polyamine binding sites in AcPhe-tRNA free in solution or bound at the P-site of Escherichia coli poly(U)programmed ribosomes. Partial nuclease digestion indicated that the deep pocket formed by nucleosides of the D-stem and the variable loop, as well as the anticodon stem, are preferable polyamine binding sites for AcPhe-tRNA in the free state. ABA-spermine was a stronger cross-linker than ANB-spermine. Both photoprobes were linked to AcPhe-tRNA with higher affinity when the latter was nonenzymatically bound to poly(U)-programmed ribosomes, In particular, the cross-linking at the T psi C stem and acceptor stem was substantially promoted, The photolabeled AcPhe-tRNA.poly(U)ribosome complex exhibited moderate reactivity towards puromycin, The attachment of photoprobes to AcPhe-tRNA was mainly responsible for this defect. A more complicated situation was revealed when the AcPhe-tRNA poly(U) ribosome complex was formed in the presence of translation factors; the reactivity towards puromycin was stimulated by irradiating such a complex in the presence of photoprobes at 50 mu M, with higher concentrations being inhibitory, The stimulatory effect was closely related with the binding of photoprobes to ribosomes, The results are discussed on the basis of possible AcPhe-tRNA conformational changes induced by the incorporation of photoprobes.