Effects of two photoreactive spermine analogues on peptide bond formation and their application for labeling proteins in Escherichia coli functional ribosomal complexes

The effect of two photoreactive analogues of spermine, N-1-azidobenzamidino - (ABA-) spermine and N-1-azidonitrobenzoyl- (ANB-) spermine, on ribosomal functions was studied in a cell-free system derived from Escherichia coli. In the dark, both analogues stimulated the binding of AcPhe-tRNA to poly(U) -programmed ribosomes, enhanced the stability of the ternary complex AcPhe- tRNA poly(U) ribosome (complex C), and caused stimulatory and inhibitory ef fects on peptidyltransferase activity. ABA-spermine exhibited more pronounc ed effects than ANB-spermine. Each photoprobe was covalently attached after irradiation to both ribosomal subunits and also to free rRNA isolated from 70S ribosomes. Photolabeled complex C showed a reactivity toward puromycin , similar to that exhibited by complex C reacting reversibly with photoprob es free in solution. The distribution of the incorporated radioactivity amo ng the ribosomal components was determined under two experimental condition s, one stimulating and the other inhibiting peptidyltransferase activity. U nder both conditions, ABA-spermine was the strongest cross-linker. Upon sti mulatory conditions, 14% of ABA-[C-14]spermine cross-linked to complex C wa s bound to the protein fraction. The proteins primarily labeled were identi fied as S3, S4, L2, L3, L6, L15, L17, and L18. Upon inhibitory conditions, a higher percent of the incorporated radioactivity was found in ribosomal p roteins, while the pattern of protein labeling was characterized by a remar kable decrease of cross-linked proteins L2, L3, L6, L15, L17. and L18 and b y an increase of cross-linked proteins S9, S18, L1, L16, L22, L23, and L27. On the basis of these results and literature data, the involvement of sper mine in the conformation and important functions of ribosomes is discussed.