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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]

It was shown that at 20 mM Mg2+ and 0 or 20 °C each 70-S ribosome binds non-enzymatically about one Phe-tRNAPhe molecule in the A site if poly(U) is used as messenger. Only a minor amount of ribosomes (never exceeding 15%) and after long incubation binds a second molecule of Phe-tRNAPhe and forms diphenylalanyl-tRNA. If the Phe-tRNAPhe preparation contains nonspecific deacylated tRNA, the latter is always bound in the P site up to one molecule per 70-S ribosome. The association constant for this non-specific tRNA binding is equal to (1.4 ± 0.3) × 107 M-1 and is independent of the messenger poly(U) and the filling of the A site by a specific aminoacyl-tRNA. It was found that the codon-dependent binding of Phe-tRNAPhe in the A site is reversible, but two different types of complexes are formed with drastic differences of binding constants. This heterogeneity is due to the presence of two conformers of Phe-tRNAPhe. The equilibrium of the two Phe-tRNAPhe conformers depends on Mg2+ and can be shifted if tRNA is heated and Mg2+ added or sequestered by complexing agents. One type of Phe-tRNAPhe yields complexes with 70-S ribosomes showing extremely high affinity constants (higher than 1011 M-1). The second conformer is characterized by a comparatively low binding constant about 5 × 106 M-1. In the binding of both species to 30-S subunits no difference is seen. A big difference in the behaviour of both Phe-tRNAPhe conformers is found also in the absence of messenger RNA. The association constant of Phe-tRNAPhe (high-affinity conformer) to 70-S ribosomes is about 3 × 108 M-1, but for the low-affinity conformer is hundred times lower. [ABSTRACT FROM AUTHOR]

Association between ribosomal subunits of Eschrichia coli can lead to the formation of two classes of 30-S · 50-S couples. The first class sedimenting between 50 S and 70 S, arises when native subunits associate, the second sedimenting at 70 S upon coupling of so-called derived subunits. From cross experiments it is concluded that it is the 50-S subunit which determines the nature of the association product, whereas the 30-S subunit determines whether an association product can be formed. Native and derived 50-S subunits are indistinguishable by sucrose gradient centrifugation and do not differ qualitatively in their ability to form polypeptides under the directions of bacteriophage RNA. Native 30-8 subunits lose a factor during isolation, probably IF-3. They are therefore able to associate both at 0° and 37 °C. On the other hand derived 30-S subunits are unable to associate at 0 °C but they do so at 37 °C in the presence of 10 mM Mg2+. A number of trivial factors have been excluded to explain the difference between the two types of 50-S subunits. The preparation method is not responsible for the difference. Nor do the derived subunits contain peptidyl-tRNA, known to influence subunit association. The content of tRNA and aminoacyl-tRNA has been determined quantitatively by polyacrylamide-gel electrophoresis. None of the subunits nor the 70-S ribosomes from which the derived subunits are prepared contain a tRNA species. [ABSTRACT FROM AUTHOR]