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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 deoxyoctanucleotide (5′-3′)d(A-A-G-G-A-G-G-T), which is complementary to the 3′ end of 16-S RNA, inhibits the formation of the complex between the 30-S subunit and MS2 RNA described in the preceding paper. If the complex is preformed, the octanucleotide cannot prevent entry of the complex into the ribosome cycle upon supplementation with the components for protein synthesis. The subunit · MS2-RNA complex is unable to bind the octanucleotide. It is concluded that in the subunit · phage-RNA initiation precursor the 16-S terminus is base-paired with a complementary MS2 RNA sequence. Edeine, aurintricarboxylic acid and antibodies against ribosomal protein S1 prevent the association of phage RNA with 30-S subunits. These compounds do not, however, inhibit the binding of (5′-3′)d(A-A-G-G-A-G-G-T) to 30-S subunits. It is concluded that the formation of the complex between MS2 RNA and 30-S subunits does not depend solely on the Shine and Dalgarno base-pairing reaction. [ABSTRACT FROM AUTHOR]

The confronmation in solution of duck and rabbit globin mRNA, and of the duck mRNA in the mRNA · protein particle, has been investigated by optical methods and also by the use of the dye ethidium bromide which becomes highly flourescent when intercalated into the double-stranded regions of a nucleic acid. On the basis of the properties of this dye and on the ability of homopolyribonucleotides to form double-stranded structures we have, in addition, developed a simple and sensitive assay for the detection and quantitisation of sequences rich in a particular residue that may be present in an RNA chain. In solutio, 45 to 60% of the nucleotides of duck globin mRNA were found to be in bihelical regions. A similar degree of secondary structure was found in rabbit globin mRNA (this paper), as well as in calf lens mRNA and mRNAs from ewe mammary gland (other results). All samples of globin mRNA examined in this work contained a sequence of poly(A), which has poly(U) binding properties similar to that of synthetic poly(A): no specific interaction between the poly(A) sequence and the rest of the molecules can be detected. The fraction of adenosine residues within these poly(A) segments represents 4% in rabbit mRNA and 8 to 9% in duck mRNA. An additional adenosine-rich segment interspersed with guanosine and possibly residues, was also detected in one duck mRNA sample. The RNA in the duck mRNA · protein particle is also highly structured. The melting profile in the range of 20 to 65° C is quite similar to that of free mRNA and the ability of ethidium bromide to intercalate is reduced to the extent of 70%. Yet the dichroïc spectra of free and bound mRNA are significantly distinct. These data suggest that free and protein-bound mRNA may have a very similar degree of secondary structure but with distinct detailed conformation in bihelical regions (change in base tilting for example). Direct evidence has been obtained that proteins stick to the poly(A) segment in the particle since the fraction of adenosine residues detectable by our poly(A) titration procedure is reduced to 50% of that observed in the free mRNA. [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]