Studies on the role of amino acid residues 31 through 46 of ribosomal protein S4 in the mechanism of 30 S ribosome assembly.

We have described previously the isolation of a large fragment of 30 S ribosomal protein S4 (Changchien &; Craven, 1976). This S4-fragment is produced by the digestion of the S4–16S RNA complex with trypsin and it retains a full capacity to associate specifically with 16S RNA. It was also demonstrated that the S4-fragment has approximately 46 amino acid residues missing from the N-terminus and an intact C-terminus (also shown by Newberry et al., 1977). Preliminary experiments with this S4-fragment indicated that it could not fully replace the intact protein S4 in the process of 30 S ribosome assembly in vitro.We have also recently reported (Changchien et al., 1978) the preparation of a new fragment of protein S4 which has only 30 amino acid residues cleaved from the N-terminus. This was achieved by the use of the reagent 2-nitro-5-thiocyanobenzoic acid which selectively modifies the cysteine residue at position 31 followed by a cleavage of the adjacent peptide bond.We have now fully characterized the capacity of these two fragments, S4-fragment (47–203) and S4-fragment(31–203), to participate in the 30 S ribosome assembly process in vitro. Using 2-dimensional polyacrylamide gel electrophoresis, we find that when S4-fragment(47–203) is a component of the in vitro assembly reaction, proteins S1, S2, S10, S18 and S21 fail to become incorporated into the final particle. In contrast, S4-fragment(31–203) appears to participate in the reconstitution reaction without impairment allowing the complete incorporation of all 20 proteins of the 30 S subunit. The resultant particle, containing the S4-fragment (31–203), is fully active in the binding of poly(U), but is completely inactive for non-enzymatic poly(U)-directed binding of Phe-tRNA (Changchien et al., 1978). These results suggest that residues 1 through 30 of protein S4 are not involved in the assembly of the 30 S ribosome, but are required for the proper construction of the tRNA binding site. In addition residues 31 through 46 must be somehow critically important for the assembly of proteins S1, S2, S10, S18 and S21. We present evidence to show that the absence of residues 31 through 46 of protein S4 prevents a conformational change in the structure of 16 S RNA which normally accompanies the RI to RI transition and that this results in the inability of these proteins to participate in the assembly process.