Ribosomal protein S15 from Escherichia coli modulates its own translation by trapping the ribosome on the mRNA initiation loading site.

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 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...

The role of mRNA structure in translational control in bacteria

“…Binding of EcS15 to the pseudoknot has been shown to trap the 30S subunit onto the mRNA in a non-productive initiation complex. 13,17 The structures of the entrapped and productive initiation complexes were recently solved by cryo-electron microscopy. 17 In the active complex, the unpaired mRNA is positioned in the classical mRNA channel and promotes the formation of the codon-anticodon interaction in the peptidyl-tRNA (P-) site.…”

“…2C). 13 Such a structural motif displays diverse functions in the regulation of protein synthesis in bacteria as well as in viruses and eukaryotes. 10 Figure 2. mRNA-protein induced-fit allows translation regulation via different mechanisms: the example of ribosomal protein S15 autoregulation in different bacteria.…”

Towards deciphering the principles underlying an mRNA recognition code

“…In Escherichia coli, S15 recognizes a pseudoknot structure folded within its own mRNA [25][26][27], rather than the three-way junction architecture associated with thermophilic bacteria. Although the ribosome can interact with mRNA already bound to S15, it cannot initiate translation in E. coli [28]. A long-awaited cryo-electron microscopy study [29] showed that in the stalled ribosome, S15 positions itself along the mRNA pseudoknot, and the S15-mRNA complex is nested on a special platform of the small ribosomal subunit (Figure 3b).…”

S6:S18 ribosomal protein complex interacts with a structural motif present in its own mRNA

“…In a number of cases their expression is regulated through a feedback mechanism, in which one or more protein products of a given operon acts as a regulator (Babitzke et al 2009). Upon saturation of the binding site on a ribosomal RNA (rRNA), a regulator-protein binds to its own mRNA, frequently within the 5 ′ untranslated region (5 ′ UTR) (Nomura et al 1980;Freedman et al 1987; Thomas and Nomura 1987;Portier et al 1990;Philippe et al 1993;Guillier et al 2002). This interaction inhibits translation initiation by occlusion of a ribosome binding site or ribosome entrapment, permitting maintenance of the balance of r-protein and rRNA levels.…”