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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 cloacin DF13, are able to enter and complete an elongation cycle. As we have shown in the preceding paper in this journal, these defective ribosomes can form 70-S initiation complexes with MS2 RNA as a messenger. Binding of the second aminoacyl-tRNA (alanyl-tRNA) to the latter complexes led to the formation of fMet-Ala-tRNA which was fully puromyein-sensitive. In addition to these aminoacyl-tRNAs also the third aminoacyl-tRNA (seryl-tRNA) can be bound to defective ribosomes. Dual-label experiments showed that puromycin released all radioactivity from the ribosomes. This indicates that fMet-Ala-Ser-tRNA is formed and is translocated from the A site to the P site. Control ribosomes bind the three aminoacyl-tRNAs in a 1:1:1 ratio. The defective particles bind fMet-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 aminoacyt-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 only four or five amino acids. The particles which temporarily survive ternary complex binding are fully capable of transpeptidation and translocation. [ABSTRACT FROM AUTHOR]

Virginiamycin S is an inhibitor of protein synthesis in vivo. In this paper we show by equilibrium dialysis that it binds specifically to the 50-S subunit of Escherichia coli ribosomes, with one binding site per subunit. This binding is not altered by the presence of chloramphenicol, tetracycline or puromycin but is competed for by erythromycin. Using the splitting-reconstitution method, it could be demonstrated that protein L16 is absolutely required for the binding of virginiamycin S to the 50-S subunit. [ABSTRACT FROM AUTHOR]

The two types of 30-S · 50-S couples described in the preceding paper, differ markedly in their sensitivity to hydrostatic pressure evoked during centrifugation in sucrose gradients. The sedimentation coefficient of about 60 S recorded with couples formed from native subunits is only apparent, the real a value being 70 S. The latter couples can be stabilized against pressure-induced dissociation by bound tRNA. On the other hand this binding renders couples formed from derived subunits more sensitive to this dissociation. As the factor determining this intriguing difference in pressure sensitivity lies in the 50-S subunit, a comparative analysis of a number of ribosomal constituents has been performed for native and derived 50-S particles. No qualitative differences in the proteins L1–L34 have been detected. Quantitative differences cannot yet be excluded. It is considered unlikely that differences in rRNAs or low-molecular-weight components arc responsible for the distinct association behaviour. Partial reconstitution of ribosomal γ-cores and split proteins detached by CsCl reveals that the determinant factor is located in the core particles. This suggests that native and derived 50-S subunits do not differ in conformation only. The possibility may be envisaged therefore, that the two types of 50-S differ in a ribosomal protein. [ABSTRACT FROM AUTHOR]