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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]

The lytic enzyme from bacteriophage T7 has been purified from T7-infected Escherichia coli B cells and characterized. In the purification procedure advantage was taken of the reversible binding of the enzyme to chitin. Molecular weight determinations of the enzyme by Sephadex G-75 chromatography and sodium dodecyl sulphate gel electrophoresis gave estimates of 21000 and 18000 respectively. Because the activity of the enzyme falls rapidly to 1-3% of that observed immediately after lysis of the cells, various means to stabilize the enzyme have been tried. Of the methods attempted only binding to the substrate analogue chitin was successful. The enzymic activity was found to be markedly dependent on ionic strength and pH, maximum activity was observed using 5 mM phosphate buffer pH 7.5. MgCl2 was found to enhance the lytic activity 2–3-fold, the optimal concentration being about 0.5 mM. The enzyme was not sensitive to penicillin G or spermine, but was specifically inhibited by p-chloromercuribenzoate. Following digestion of E. coli peptidoglycan the cell wall peptides L-Ala-D-Glu(A2pm), L-Ala-D-Glu(A2pm-D-Ala) and the dimer of the latter were formed, together with paper chromatographically immobile glycan strands. The enzyme, therefore, acts as an N-acetylmuramyl-L-alanine amidase by cleaving the bond between N-acetylmuramic acid and L-alanine in the E. coli peptidoglycan. The enzyme has apparently no effect on the peptidoglycan from Micrococcus luteus, and seems to have a preference for high-molecular-weight substrates. Thus the disaccharide-tetrapeptide GlcNAc-MurNAc-L-Ala-D-Glu(A2pm-D-Ala) and its dimer are not substrates for the enzyme. [ABSTRACT FROM AUTHOR]