J. Mol. Biol. (lY88) 200, 309-319 Probing the Assembly of the 3’ Major Domain of 16 S Ribosomal RNA Quaternary Interactions Involving Ribosomal Proteins S7, S9 and S19 Ted Powers’, L&Ming Changchien 2, Gary R. Craven2 and Harry F. Noller’ 1Thimann Laboratories University of California at Santa Cruz Santa Cruz, CA 95064, U.S.A. 2Laboratory of Molecular Biology University of Wisconsin Madison, WI 53706, U.S.A. (Received 26 May 1987) We have studied the effect of assembly of ribosomal proteins 57, S9 and S19 on the accessibility and conformation of nucleotides in 16 S ribosomal RNA. Complexes formed between 16 S rRNA and S7, S7+S9, S7+Sl9 or S7+S9+S19 were subjected to a combination of chemical and enzymatic probes, whose sites of attack in 16 S rRNA were identified by primer extension. The results of this study show that: (1) Protein S7 affects the reactivity of an extensive region in the lower half of the 3’ major domain. Inclusion of proteins S9 or S19 with S7 has generally little additional effect on S7specific protection of the RNA. Clusters of nucleotides that are protected by protein S7 are localized in the 935-945 region, the 950/1230 stem, the 1250/1285 internal loop, and the 1350/1370 stem. (2) Addition of protein S9 in the presence of S7 causes several additional effects principally in two structurally distal regions. We observe strong SS-dependent protection of positions 1278 to 1283, and of several positions in the 1125/1145 internal 190~. These findings suggest that interaction of protein S9 with 16 S rRNA results in a structure in which the 1125/1145 and 1280 regions are proximal to each other. (3) Most of the strong SlS-dependent effects are clustered in the 950-1050 and 121CL1230 regions, which are joined by base-pairing in the 16 S rRNA secondary structure. The highly conserved 960-975 stemploop, which has been implicated in tRNA binding, appears t)o be destabilized in the presence of S19. (4) Protein S7 causes enhanced reactivity at several sites that become protected upon addition of S9 or S19. This suggests that S7-induced conformational changes in 16 S rRNA play a role in the co-operativity of assembly of the 3’ major domain. 1. Introduction the 3’ maior domain was excised from naked 16 S Early evidence for the domain organization of rRNA (Zimmermann et al., 1972) or as an RNP ribosomal RNA came from partial nuclease containing a subset of the ribosomal proteins from digestion studies of 16 S rRNA and 30 S subunits. 30 S subunits (Brimacombe et al., 1971; Yuki & In retrospect, the results of several such Brimacombe, 1975), by partial nuclease digestion. experiments provide rather convincing evidence RNPs isolated by two other groups (Schendel et al., that the 3’ major domain of 16 S rRNA (as defined 1972; Roth & Nierhaus, 1973) are probably related by Noller & Woese, 1981), in particular, forms a to the one that was characterized by Brimacombe relatively autonomous ribonucleoprotein (RNPt) and co-workers (Morgan & Brimacombe, 1972; Yuki structure within the assembled 30 S subunit. & Brimacombe, 1975); in all cases, proteins S7, S9 A subfragment corresponding almost precisely to and S19 were found, usually in near-stoichiometric amounts, together with varying amounts of other proteins. Assembly experiments in vitro showed t Abbreviations used: RIVP, ribonucleoprotein; DMS, that protein S7 binds independently to 16 S rRNA dimethylsulfate; u.v., ultraviolet. (Schaup et al., 1970; Mizushima & Nomura, 1970; 309 0022%2836/88/06030~11 $03.00/O 0 1988 .Amdemic Press Limited