RIBOSOMAL-PROTEIN S15 FROM ESCHERICHIA-COLI MODULATES ITS OWN TRANSLATION BY TRAPPING THE RIBOSOME ON THE MESSENGER-RNA INITIATION LOADING SITE

From genetic and biochemical evidence, we previously proposed that S15 inhibits its own translation by binding to its mRNA in a region overl apping the ribosome loading site. This binding was postulated to stabi lize a pseudoknot structure that exists in equilibrium with two stem-l oops. Here, we use ''toeprint'' experiments with Moloney murine leukem ia virus reverse transcriptase to analyze the effect of S15 on the for mation of the ternary mRNA-30S-tRNA(f)Met complex. We show that the bi nding of the 30S subunit on the mRNA stops reverse transcriptase near position +10, corresponding to the 3' terminus of the pseudoknot, most likely by stabilizing the pseudoknot conformation. Furthermore, S15 i s found to stabilize the binary 30S-mRNA complex. When the ternary 30S -mRNA-tRNA(f)Met 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 T1 footprinting experiments reveal the simultaneous binding of S15 and 3 0S subunit on the mRNA. Otherwise, we show by filter binding assays th at initiator tRNA remains bound to the 30S subunit even in the presenc e of S15. Our results indicate that S15 prevents the formation of a fu nctional ternary 30S-mRNA-tRNA(f)Met complex, the ribosome being trapp ed in a preternary 30S-mRNA-tRNA(f)Met complex.