Of the three proteins, L5, L18 and L25, which bind to 5S RNA, the former two effect the interaction of 5S RNA with 23S RNA. We have used trypsin as a probe to investigate the roles of the proteins in this RNA-RNA assembly, with the following results:(1) In complexes with 5S RNA, the highly basic N-terminal region of L18 is accessible to trypsin. This accessibility is unaffected by L25. However, its presence is essential for stimulating L5 binding. (2) In 5S RNA-protein-23S RNA complexes proteins L5 and L18 are both strongly resistant to proteolysis. (3) No 5S RNA-23S RNA complex formation occurs in the presence of L5 and the C-terminal L18 fragment. Two possible models for the mechanism of RNA-RNA assembly are proposed.

A fragment of ribosomal protein S4 was prepared by limited trypsin digestion of a specific complex between protein S4 and 16-S RNA. It was characterised for amino acid sequence and the N-terminal 46 amino acids were found to be absent. An intermediate fragment, cut at Arg-43, was also observed at low trypsin concentrations. Evidence is presented that the protected fragment constitutes the primary RNA-binding region of the protein. No smaller protein fragments were found that rebound to the RNA. A mechanism for the degradation of the N-terminal region of the protein is proposed and two probable functions of the excised region are given.Under milder trypsin digestion conditions than for the complex, the same fragment, cut at Arg-46, was also prepared from the free protein. This result, together with that from a control experiment, indicates that at least within this local region, the protein conformation is conserved in both the free protein and the protein . RNA complex. This is the first direct evidence for the conservation of conformation in a protein when both complexed and uncomplexed with a ribosomal RNA.Protein S4 has an important role in the assembly in vitro of the 30-S ribosomal subunit of Escherichia coli [l]. Moreover, it binds directly and specifically to a large RNA region at the 5'-end of the 16-S RNA that is extensively folded into a tertiary structure [2,3].Hydrodynamic studies [4], low-angle X-ray scattering studies [5,6] and the electron microscopic visualisation of ribosome-bound antibody markers [7,8] all indicate that the protein has a highly asymmetric structure. Furthermore, nuclear magnetic resonance and other spectroscopic studies [9,10] have indicated that the protein has an open and extended structure with about 30 % a-helix and little p-structure.In an earlier report we demonstrated that on trypsin digestion of complexes between protein S4 and 16-S RNA a large protein fragment was protected by the RNA against digestion and that this was able to rebind to the RNA [ll]. We believe that this limited proteolysis approach may be of general application to identifying primary RNA binding regions of ribosomal proteins. In the present study the protected S4 fragment has been characterised for amino acid sequence. It has been shown, unequivocably, that the fragment binds specifically to the 16-S RNA. Moreover, we find that the same protein fragment can be isolated from the free protein indicating some conservation of structure in the free and in the RNAbound protein.

There are the following minor alterations to Figs 2 and 3. (i) Peptide T8 is incorrectly assigned on the peptide map; the spot is an incompletely digested basic peptide. This does not affect the conclusion that the L1 8 fragment terminates at Lys-l 7 and Leu-1 8. (ii) Peptide T12 has only been tentatively assigned at the origin of the peptide map. (iii) The incomplete trypsin digestion products denoted in Fig.Z as T3a, T4a etc. are not shown in Fig. 3. They represent adjacent tryptic peptides, as shown for T8a. Thus, for example, T3a is T3 + T4. Other LI 8 sequence details are only available in the

1. Evidence is presented for the occurrence of a very stable RNA core (S4-RNA) in "native" 16S RNA that is also present in the 30S subunit of Escherichia coli. A model giving the approximate location of this RNA core in the 30S subunit is presented. 2. It is proposed (a) that this S4-RNA acts as a nucleus for the assembly of the 30S subunit, and (b) that a small class of "linkage" proteins, including S4, further facilitate the assembly of the proteins to the RNA, thereby explaining some of the "cooperative" effects that are observed during in vitro assembly. 3. Evidence for the importance of the RNA core in the functioning of the ribosome is discussed.

No abstract