Consequences of a specific Cleavage in situ of 16-S Ribosomal RNA for polypeptide Chain Elongiation

In this paper the question was studied whether Escherichia coli ribosomes which have undergone a specific cleavage of their 16-S rRNA by treatment with the bacteriocin cloacn DF13, are able to enter and complete an elongation cylce. As we shwon in the preceding paper in this journal, these defective ribosomes can form 70-S initiation complexes wiht MS2 RNA as a messnger. Binding of the second aminocyl-tRNA (alanl-tRNA) to the latter complexes led to the foramtion of fMet-Ala-rRNA which was fully puromycin-sensitive. In addition to these aminocacyl-tRNA also the third aminoacyl-tRNA (serl-tRNA) can be bound to defective ribosomes. Dual-label experiments showed that puromycon relaeased all radiactivity from the ribosomes. This inadicated that fMet-Ala-Ser-tRNA is formed and is translaocated from the A site to the P site. Control ribosomes bind the three aminoacyl-tRNAs in 1:1:1 ratio. The defective particles bind fMet-Ala-tRNA, Ala-tRNA and Ser-tRNA in a ratio of 1:0:5:0.25. Kinetic experiments strongly suggest that at each binding of the ternary complex aminoacyl-tRNA · EF-Tu · GTP to the A site of defective ribosomes, about half of these particles undergo irreversible inactivation. Consequently polypeptide synthesis under our conditions will come to an end after incorporation of ony four or finve amino acids. The particles which temporarily survive thernary complex binding are fully capalbe of transpeptidation and tanslocation.