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When Escherichia coli 30-S ribosomal subunits are hydrolysed under mild conditions, two ribonucleoprotein fragments of unequal size are produced. Knowledge of the RNA sequences contained in these hydrolysis products was required for the experiments described in the preceding paper, and the RNA sub-fragments have therefore been examined by oligonucleotide analysis. Two well-defined small fragments of free RNA, produced concomitantly with the ribonucleoprotein fragments, were also analysed. The larger ribonucleoprotein fragment, containing predominantly proteins S4, S5, S8, S15, S16 (17) and S20, contains a complex mixture of RNA sub-fragments varying from about 100 to 800 nucleotides in length. All these fragments arose from the 5′-terminal 900 nucleotides of 16-S RNA, corresponding to the well-known 12-S fragment. No long-range interactions could be detected within this RNA region in these experiments. The RNA from the smaller ribonucleoprotein fragment (containing proteins S7, S9, S10, S14 and S19) has been described in detail previously, and consists of about 450 nucleotides near the 3′ end of the 16-S RNA, but lacking the 3′-terminal 150 nucleotides. The two small free RNA fragments (above) partly account for these missing 150 nucleotides; both fragments arose from section A of the 16-S RNA, but section J (the 3′-terminal 50 nucleotides) was not found. This result suggests that the 3′ region of 16-S RNA is not involved in stable interactions with protein. [ABSTRACT FROM AUTHOR]

A ribonucleoprotein prepared by strong ribonuclease digestion of a complex of 16-S ribosomal RNA and proteins S4 and S20 from Escherichia coli has been characterized; its nucleotide sequence, the positions of enzyme cuts and the sequence excisions have been placed in the completed sequence of 16-S RNA. The positions and yields of enzyme cuts, and excisions of sequence, are compared with those of various ribonucleoproteins prepared with S4 or S20 alone, and with the ribonuclease-resistant S4 RNA prepared from renatured 16-S RNA in the absence of ribosomal protein. These data yield important information on the topography and organization of the 5′ third of the 16-S RNA which is selectively maintained in its native conformation by the bound proteins; they also provide criteria for testing secondary structural models of this region of 16-S RNA. [ABSTRACT FROM AUTHOR]

32-P-labelled 50-S subunits from Escherichia coli ribosomes were hydrolysed under conditions known to give rise to two specific ribonucleoprotein fragments, containing proteins L1, L9, and L5, L18 and L25 respectively. RNA corresponding to these ribonucleoproteins was isolated and purified, and the various RNA fragments obtained were subjected to oligonucleotide analysis. The results showed that the RNA associated with proteins L1 and L9 was very similar to the RNA found with protein L1 after controlled digestion of 23-S-RNA · L1 complexes (described elsewhere); this RNA lies within a region 550–1000 nucleotides from the 3′ terminus of 23-S RNA. The RNA associated with proteins L5, L18, and L25 consisted predominantly of two species of similar size. One was 5-S RNA, and the other a fragment of 23-S RNA, lying within the region 450–1000 nucleotides from the 3′ terminus. [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]

1. 30-S ribosomal subparticles were hydrolysed with ribonuclease T1 or pancreatic ribonuclease in the presence of 2 M urea. The RNA · protein fragments produced were separated on 5% polyacrylamide/0.50% agarose composite gels. Fractions from the composite gels were subjected to protein analysis on 17.5% periodate-soluble polyacrylamide gels run in the detergent sarkosyl, using the technique already published. 2. Several RNA · protein fragments were obtained by this procedure, each containing equimolar amounts of a few specific ribosomal proteins. Strict criteria for specificity of the proteins in each fragment were applied. Proteins S8 and S15 were found reproducibly in a small RNA · protein fragment, with only trace amounts of the remaining ribosomal proteins. Six proteins (S7, S9, S10, S13, S14, and S19) constitute another group, and fragments containing three, four, five, or all six of these proteins were observed. Protein S20 was occasionally associated with this group, or with S6, S16 or S17, and possibly S21. The latter (including S20) were also found in association with S8 and S15. 3. The individual proteins found in specific fragments are interpreted as being close neighbours in the 30-S particle. The fragment data can be incorporated into a re-arranged version of Nomura's "assembly map". [ABSTRACT FROM AUTHOR]