From genetic and biochemical evidence, we previously proposed that S15 inhibits its own translation by binding to its mRNA in a region overlapping the ribosome loading site. This binding was postulated to stabilize a pseudoknot structure that exists in equilibrium with two stemloops. Here, we use "toeprint" experiments with Moloney murine leukemia virus reverse transcriptase to analyze the effect of S15 on the formation of the ternary mRNA-30S-tRNAmIet complex. We show that the binding of the 30S subunit on the mRNA stops reverse transcriptase near position + 10, corresponding to the 3' terminus ofthe pseudoknot, most likely by stabilizing the pseudoknot conformation. Furthermore, S15 is found to stabilize the binary 30S-mRNA complex. When the ternary 30S-mRNA-tRNAmet complex is formed, a toeprint is observed at position + 17. This toeprint progressively disappears when the ternary complex is formed in the presence of increasing concentrations of S15, while a shift from position + 17 to position + 10 is observed. Beside, RNase Ti footprinting experiments reveal the sinultaneous binding of S15 and 30S subunit on the mRNA. Otherwise, we show by fiter binding assays that initiator tRNA remains bound to the 30S subunit even in the presence of S15. Our results indicate that S15 prevents the formation of a functional ternary 30S-mRNAtRNAfmet complex, the ribosome being trapped in a preternary 30S-mRNA-tRNAfm complex.A number of prokaryotic and phage RNA-binding proteins are controlled by a translational feedback mechanism that allows modulation of the protein synthesis rate with respect to the intracellular concentration of its substrate RNA (for reviews, see refs. 1-4). It is commonly assumed that the regulatory mechanism proceeds through the binding of the repressor protein to the mRNA, in a target region generally near or overlapping the ribosome loading site. Up to now, regulation was believed to proceed through a simple mechanism of competition between the repressor and the ribosome. Such a mechanism has been experimentally supported for Escherichia coli threonyl-tRNA synthetase, which was shown to prevent the formation of the temary 30S-mRNAtRNAf et complex (5) and the binary 30S-mRNA complex (P. Romby, personal communication). An alternative repression mechanism has been postulated by Draper (3) in which the repressor traps the ribosome on its initiation site and prevents further elongation steps. However, no direct evidence has been provided yet for the existence of such a mechanism.The expression ofE. coli ribosomal protein S15 was shown to be negatively controlled at the translational level by a feedback mechanism and the regulatory site was located in the leader of the mRNA overlapping the ribosome loading site and the first codons (6). We have shown (7) that the regulatory region folds into three domains (Fig. 1). The first and second domains in the 5' part of the mRNA leader correspond to very stable stem-loop structures. The third domain can fold into two alternative conformations. One correspond...

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Escherichia coli ribosomal protein S8 was previously shown to bind a 16s rRNA fragment (nucleotides 584-756) with the same affinity as the complete 16s rRNA, and to shield an irregular helical region (region C) [Mougel, M., Eyermann, F., Westhof, E., Romby, P., Expert-Bezanqon, Ebel, J. P., Ehresmann, B. & Ehresmann, C. (1987). J. Mol. Biol. 198, 91-1071. Region C was postulated to display characteristic features : three bulged adenines (A595, A640 and A642), a noncanonical U598-U641 pair surrounded by two G . C pairs. In order to delineate the minimal RNA binding site, deletions were introduced by site-directed mutagenesis and short RNA fragments were synthesized. Their ability to bind S8 was assayed by filter binding. Our results show that the RNA binding site can be restricted to a short helical stem (588-609633-6.51) containing region C . The second part of the work focused on region C and on the role of conserved nucleotides as potential determinants of S8 recognition. Single and double mutations were introduced by site-directed mutagenesis in fragment 584-756, and their effect on S8 binding was measured. It was found that the three bulged positions are essential and that adenines are required at positions 640 and 642. U598 is also crucial and the highly conserved G597 . C643 pair cannot be inverted. These conserved nucleotides are either directly involved in the recognition process as direct contacts or required to maintain a specific conformation. The strong evolutionary pressure and the small number of positive mutants stress the high stringency of the recognition process.RNA-binding proteins recognize defined parts of RNA molecules in a highly specific manner, but in most cases the basic molecular mechanism of this recognition process remains not well known. Ribosomal components offer a valuable system for the possible dissection of the underlying principles of specific protein-RNA interactions. The Escherichia coli 30s subunit is composed of 21 proteins and some of them, named primary proteins, interact independently and specifically with selected regions of ribosomal 16s rRNA. Among them, protein S8 plays a central role both in 30s ribosomal subunit assembly (Held et al., 1974) and in the regulation of the Spc ribosomal protein operon (Yates et al., 1980: Dean et al., 1981. During 30s assembly, S8 binds to the central domain of 16s rRNA in the early stage of the process (Held et al., 1974) and may interact cooperatively with two other small subunit proteins: S6 and S38 Svensson et al., 1988). It was suggested that the region near S8, in the body of 30s particle, represents one of the two nucleation sites during assembly (Moore, 1987). A variety of methods, including partial nuclease digestion (Zimmermann et al., 1975;Ungewickell et al., 1975; Zim- (Mougel et al., 1987;Svensson et al., 1988) and sitedirected mutagenesis Gregory et al., 1988) was used to localize the region within 16s rRNA that mediates S8 recognition. In a previous work, we characterized the interactions between protein S8 and 16s rRN...