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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 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]

The effect of bacteriocin (cloacin DF13) treatment of Escherichia coli ribosomes on initiation of protein synthesis has been studied in detail. In agreement with out previous findings [Baan et al. (1976) Proc. Natl Acad. Sci. U.S.A, 73, 702–706] it is shown that 70-S initiation complexes can be formed with cloacin-treated ribosomes, but that the initiation factor IF-1 does not function properly. The following pleiotropic effects of this factor have been studied: (a) the acceleration of ribosomal subunit exchange with 70-S couples; (b) the stimulation of the IF-3-mediated dissociation of 70-S ribosomes; (c) the stimulation of 30-S initiation complex formation; (d) the enhancement of the rate of release of IF-2 from 70-S initiation complexes. The effects (a) and (b) are virtually abolished after cleavage of 16-S rRNA. The effect (d) is only partially reduced whereas effect (c) seems to be unimpaired. It is concluded that 70-S initiation complex formation with cloacin-treated ribosomes suffers from improper functioning of IF-1 in the generation of active subunits from 70-S tight couples. This is the only effect on initiation. It can he compensated for by adding more IF-3. The data provide functional evidence that 16-S rRNA is involved in ribosomal subnnit interaction. [ABSTRACT FROM AUTHOR]

Escherichia coli ribosomal protein S1 and its mutant, shorter, form m1-S1 were cleaved at internal methionyl residues to yield, respectively, six and five fragments of M1 ranging from 1000 to 24000. Methods are described to isolate the fragments in pure form. Four of the fragments (designated F2a, F2b, F3 and F4) contain between 86 and 215 amino acids and are therefore as large as other ribosomal proteins. Fragment F2a, derived from the N-terminal region, has previously been shown to contain the major ribosome binding domain of S1 [S. Giorginis and A. R. Subramanian (1980) J. Mot Biol. 1411 393 -408]. Here we show that the RNA binding domain of S1 is essentially contained in F3 derived from the middle region of S1 and carrying the nonreactive - SH group. The reactive a SF! group of SI, whose activity is modified by ligand binding, was localized in F2b, a fragment with little RNA binding capacity. The characteristic RNA binding domain and a weak ribosome binding domain of S1 have previously been localized in the large trypsin-resistent core SI-Fl [T. Suryanarayana and A. It Subramanian(1979) J. Mol. Biol. 127, 41-54]. Together these data indicate that two of the key functional domains of SI are located in two regions of the molecule separated by an open, exposed segment. The present study also revealed that the nonreactive - SM group of S1 becomes reactive in m1-S1 by the loss of the remote C-terminal region in the latter. [ABSTRACT FROM AUTHOR]

Analysis of various RNA species from Escherichia coli. The RNAs were prepared under mild conditions without spreading using the shadow-cast technique. The 16-S RNA looks like a flattened balloon of diameter 18 ± 1.9 nm with a smooth surface and regular contours. In contrast, structural domains can be detected in 23-S RNA; typically a central domain is surrounded by five to seven satellite domains, with an overall diameter of 33 ±_ 4.5 nm. Different 23-S RNA preparations, which also behave differently in reconstitution assays, differ with respect to extent and height of the satellite structure. 5-S RNA shows an extended rod-like structure with a length of 11 ± 1.6 nm. A more corpuscular and prominent structure was observed for tRNA, with diameters Phe of 7.7 ± 1.1 nm and 8.9 ± 1.0 nm in case of tRNAPhe, and N-acetylphenylalanyl-tRNAPhe respectively. Analysis of the assembly in vitro of the ribosomal 50-S subunit. The assembly of the 50-S subunit in vitro occurs via three reconstitution intermediates, which are formed subsequently during the first incubation of the two-step reconstitution procedure [Dohme, F. and Nierhaus, K. H. (1976) J. Mol. Biol. 107, 585-599]. A total reconstitution was performed, aliquots were withdrawn during the first incubation at various times and analyzed in the electron microscope. A second aliquot was taken at the same times, subjected to a standard second incubation and tested for poly-(Phe)-synthesizing activity in the presence of 30-S subunits. The activity obtained is a measure of the amount of (second plus third) reconsitution intermediates present in the aliquot. With this parameter it was possible to select a series of subsequent assembly states from the electron micrographs which cinematographically illustrate the assembly of the 50-S subunit in vitro. The results indicate that the reconstitution intermediate stage RI[IPQ]50(1) comprises various particles, which evidently are formed consecutively. In contrast, the following reconstitution intermediate stage RI50(2) appears as a homogeneous population of particles. [ABSTRACT FROM AUTHOR]

Substantial evidence is provided to corroborate our previous finding that Escherichia coli ribosomes recognize two binding sites on the 5′ end of alfalfa mosaic virus (AMV) RNA 4 [for a preliminary report see Castel, A., Kraal, B., Kerklaan, P. R. M., Klok, J., and Bosch, L. (1977) Proc. Natl Acad. Sci. U.S,A. 74, 5509–5513]. Translation can start at either site using AcPhe-tRNA or fMet-tRNA as initiator and takes place in the same reading frame along the monocistronic mRNA. The size and composition of the isolated extra NH2-terminal fragment of the acetylphenylalanyl product were found to be in agreement with the 5′ non-coding region of the messenger. Removal of the 5′-terminal cap structure of AMV RNA 4 did not influence significantly both initiation reactions. Ribosomal protein S1 was essential for binding as well as incorporation of both fMet-tRNA and AcPhe-tRNA. A similar interaction on the ribosome was found for AcPhe-tRNA directed by AMV RNA 4 as for fMet-tRNA directed by either AMV RNA 4 or MS2 RNA with respect to the influence of initiation factors. It is concluded that the heterologous plant viral messenger is reliably translated in the E. coli system and that E, coli ribosomes recognize with high specificity an extra initiation site close to the 5′ extremity of the messenger. The relation- ship of this site to a hypothetical entry site involved in the early recognition in the initiation mechanism between ribosome and messenger is discussed. [ABSTRACT FROM AUTHOR]

The poly(U)-dependent binding of Tyr-tRNATyr to Escherichia coli ribosomes has been studied using a highly purified system. Binding is maximal at 10 mM magnesium acetate (up to 0.7 molecule Tyr-tRNATyr/ribosome), and requires the presence of elongation factor (EF) T (a mixture of EF-Ts and EF-Tu), GTP, NH4+ ions and an aminoglycoside antibiotic (streptomycin, neomycin B, kanamycip B or gentamicin C1a). Under limiting and up to saturating concentrations of EF-T, one molecule of GTP is hydrolyzed per molecule of Tyr-tRNATyr bound, suggesting that ‘proof-reading’ mechanisms involving the hydrolysis of GTP are inoperative in the presence of the antibiotics. Binding of Tyr-tRNATyr apparently takes place at the ribosomal acceptor site, since peptide bonds are readily formed with N-acetyl-Phe-tRNA prebound to the ribosomal donor site. In contrast to Phe-tRNAPhe binding, Tyr-tRNATyr binding is impaired by the omission of the 50-S subunit, the replacement of GTP by its non-hydrolyzable analogs guanyl-5′-yl methylene diphosphonate and guanyl-5′-yl iminodiphosphonate, and also by the presence of the antibiotic streptogramin A. This suggests that the correct interaction of Tyr-tRNATyr with the peptidyl transferase centre is essential for the stability of this ligand on the ribosome. Moreover, the aminoglycoside antibiotics are also necessary, even after the binding reaction is complete, to maintain Tyr-tRNATyr on the ribosome. [ABSTRACT FROM AUTHOR]

The 3'-terminal sequences of 16-S ribosomal RNA from a number of bacteria have been determined by a stepwise degradation and 3'-terminal labelling procedure. The sequence obtained were: Bacillus stearothermophilus, -G(Z)≈5 Y-U-C-C-U-U-U-C-U (A); B subtilis, -G(Z)≈7 Y-C-U-U-U-C-U; Caulobacter crescentus, -G(Z)3 Y-U-C-C-U-U-U-C-U; Pseudomonas aeruginosa, -G-Z-Z-Y-C-U-C-U-C-C-U-U(A), where Z is any nucleotide other than G. Thus, as previously found in Escherichia coli, all bacterial 16-S rRNAs contain a pyrimidine-rich tract at the 3'-terminus. In B. stearothermophilus and Ps. aeruginosa this region shows substantial heterogeneity involving the 3'-terminal adenylic acid. A low level of 3'-terminal heterogeneity cannot be excluded for the other bacterial 16-S rRNAs examined. The 3'-termini of bacterial 16-S rRNA can be divided into two groups on the basis of sequence homology. The first group comprises E. coli and Ps. aeruginosa; the second, B. stearothermophilus, B. subtilis and C. crescentus. This division correlates with a previous separation of bacterial ribosomes into two categories based on ability to translate different mRNA preparations [Stallcup, Sharrock & Rabinowitz (1974) Biochem. Biophys. Res. Commun. 58, 92-98]. We have previously proposed that the precise base sequence at the 3'-terminus of 16-S rRNA determines the intrinsic capacity of bacterial ribosomes to translate a particular cistron [Shine & Dalgarno (1975) Nature (Lond.) 254, 34-38]. No difference was found in the 3'-terminal heptanucleotide sequence of 16-S rRNA from bacgteriophage T7-infected E. coli, as compared to that in uninfected cells. Thus, the T7-induced alteration in translational specificity of E. coli ribosomes is probably not mediated by modification of the terminal seven nucleotides of the smaller rRNA. The 3'-terminal sequences of the 23-S rRNA species were also determined. The sequences obtained were: B. stearothermophilus and B. subtilis, -Y-C; C. cresentus, -Y-C-U; Ps. aeruginosa, -Y-C-A; E. coli, -G-Y-U-U-A-A-C-C-U-U. No evidence for 3'-terminal heterogeneity was found. The results obtained are discussed in relation to possible base-pairing roles for the 3'-end of 16-S rRNA in bacterial protein synthesis. [ABSTRACT FROM AUTHOR]

16-S RNA and five pure ribosomal proteins (S3, S4, S6, S7 and S8) from Escherichia coli 30-S subunits were studied by circular dichroism in regard to the reconstitution of biologically active subunits. A small structural variation upon heating could be measured for RNA. It cannot be interpreted in terms of stacking or disruption of hydrogen bonds. However, most proteins underwent important structural modifications when heated to 42 °C in reconstitution solvent. This effect was not dependent on the presence of magnesium salt. The relative amounts of secondary structure of native and heat-denatured ribosomal proteins were calculated by using three reference curves obtained from standard proteins, the exact secondary structure of which has been determined by X-ray crystallography. In contrast with earlier results, about 18 %, β-structure was detected in native proteins. This discrepancy can most probably be attributed to the calculation method used. Results are discussed in relation to reconstitution of ribosomes. [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]

A systematic study has been made of the dissociation into subunits of the ribosomes of Escherichia coli. By analytical ultracentrifugation, ribosomes were shown to be in dynamic reversible equilibrium with their subunits. This enabled a thermodynamic analysis to be made of the association-dissociation reaction and of several factors which influence it: the concentration of monovalent and divalent cations, the presence of transfer RNA and supernatant factors, the pH, and hydrostatic pressure. The apparent change in free energy for the association in 10 mM sodium phosphate pH 7.0 was -17.4 kcal/mol. Association was accompanied by the net binding of magnesium ions or the net release of monovalent cations (potassium, sodium, or Tris ions). Circular dichroism measurements of ribosomes and their isolated subunits over a range of magnesium and potassium ion concentrations indicated that a change in the conformation of each subunit accompanied association. The change occurring in the 50-S subunit was dependent on the presence of potassium ions, whereas the change in the 30-S subunit was magnesium-ion dependent. The conformational change in the 50-S subunit was critical for the association-dissociation reaction. Under ionic conditions which lead to ribosome dissociation one molecule of transfer RNA was released per 70-S ribosome. This was an independent reaction which did not influence the subunit association-dissociation equilibrium. The release of transfer RNA was accompanied by the net release of four magnesium ions, and occurred with an apparent change in the free energy of 20.8 kcal/mol. [ABSTRACT FROM AUTHOR]