Abstract
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 stem-loops. 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-tRNA(fMet) complex. We show that the binding 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 is found to stabilize the binary 30S-mRNA complex. When the ternary 30S-mRNA-tRNA(fMet) 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 30S subunit on the mRNA. Otherwise, we show by filter 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-mRNA-tRNA(fMet) complex, the ribosome being trapped in a preternary 30S-mRNA-tRNA(fMet) complex.