PEPTIDE ENVIRONMENT OF THE PEPTIDYL TRANSFERASE CENTER FROM ESCHERICHIA-COLI 70-S RIBOSOMES AS DETERMINED BY THERMOAFFINITY LABELING WITH DIHYDROSPIRAMYCIN

In an attempt to gain information about the peptidyl transferase center at the peptide level we cross-linked the spiramycin derivative dihydrospiramycin to its functional binding site in the 70 S ribosome of Escherichia coli, In this manner ribosomal proteins S12, S14, L17, L18, L27 and L35 were found specifically affinity-labeled, Proteolytic fragmentation of these proteins, separation by C-18 reversed-phase high performance liquid chromatography of the peptide mixtures, and subsequent sequence analysis of labeled peptides revealed peptide regions at positions Ala(1)-Lys(9) and Tyr(116)-Lys(119) of S12, Leu(47)-Asp(53) of protein S14, Ser(6)-Lys(35) of protein L17, Ala(57)-Lys(63) of protein L18, Ala(5)-Lys(18) and Val(66)- Lys(71) of protein L27, and Thr(5)-Lys(11) of protein L35. This approach is a valuable tool to characterize the binding site of spiramycin as well as the peptidyl transferase center at the molecular level.