Identification in a pseudoknot of a UzG motif essential for the regulation of the expression of ribosomal protein S15 (pseudoknotyUzG base pairyRNA binding)

The ribosomal protein S15 from Escherichia coli binds to a pseudoknot in its own messenger. This interaction is an essential step in the mechanism of S15 translational autoregulation. In a previous study, a recognition determinant for S15 autoregulation, involving a UzG wobble pair, was located in the center of stem I of the pseudoknot. In this study, an extensive mutagenesis analysis has been conducted in and around this UzG pair by comparing the effects of these mutations on the expression level of S15. The results show that the UzG wobble pair cannot be substituted by AzG, CzA, AzC, GzU, or CzG without loss of the autocontrol. In addition, the base pair CzG, adjacent to the 5* side of U, cannot be f lipped or changed to another complementary base pair without also inducing derepression of translation. A unique motif, made of only two adjacent base pairs, UzGyCzG, is essential for S15 autoregulation and is presumably involved in direct recognition by the S15 protein. Although RNA pseudoknots have been implicated in many different regulatory functions (1–8), very little is known about how proteins specifically recognize these structures. In one of the most studied cases, ribosomal frameshifting, protein–RNA recognition studies are difficult because the pseudoknot interacts with a complex protein-synthesizing machinery. A more favorable case is that of the autoregulation of ribosomal protein S15 from Escherichia coli. When S15 (10 kDa) is in excess of that required for ribosome assembly, it binds specifically to a pseudoknot structure that forms transiently around the ribosome loading site of its own messenger, thus repressing its own synthesis (9–11). The secondary structure of the pseudoknot was determined in vitro by enzymatic and chemical probing of S15 mRNA fragments (12, 13). These studies showed that the pseudoknot is unstable and is in equilibrium with another form consisting of two stem-loops. In vitro, S15 binds only to the pseudoknot, as shown by the tight correlation between S15-specific mRNA binding and the ability of the bound RNA to form a pseudoknot. Protection experiments with chemical probes identified two regions shielded by S15 in the pseudoknot (black dots in Fig. 1): one located at the distal end of stem 2 and covering loop 1 and the other in stem 1 near the junction of the two stems (13), suggesting, at first glance, that two contact areas in the minor groove might be involved. Protection by S15 against hydroxyl radical attack is restricted to the interaction essentially around the coaxial stack of the two stems (13). In all cases, the bases protected include the U of a U(249)zG(236) wobble base pair present in stem 1. In vivo, evidence for pseudoknot formation was obtained by analyzing the effects of compensatory mutations on the regulatory properties of a translational fusion between rpsO, the S15 gene, and lacZ (10). An extensive mutagenesis of stem 2 failed to reveal any base involved in autoregulation or S15 binding, suggesting that putative contacts in this region identified by the chemical protection experiments might be unspecific or associated to functional groups belonging to the backbone. On the other hand, mutational analysis of stem 1 showed that a recognition determinant is located in this stem and involves a UzG wobble pair (10). Several examples are known where GzU pairs are involved in RNA recognition (14–17). However, direct recognition of this pair has not been clearly established because it was generally possible (at least in vivo) to substitute the wobble pair by some other mispairs, suggesting that access to the functional groups is controlled by the helix geometry. Here it is shown that, in vivo, UzG cannot be substituted by other canonical or noncanonical base pairs and that UzG is included in a motif that probably determines a unique configuration of the wobble pair. MATERIALS AND METHODS Mutagenesis. Mutations were introduced into the pseudoknot by site-directed mutagenesis (18, 19) of M13 mp8 derivatives carrying a translational fusion between rpsO and lacZ (10). After mutagenesis, an EcoRI–HindIII fragment carrying all the regulatory region was sequenced and fused in frame to the distal part of lacZ, which is carried by a l derivative phage (9). Encapsidation, infection of the E. coli strain AB5321 (argG, argE, his, rpsL, DlacX74), and plaque screening were carried out as previously described (9, 10). Monolysogens were isolated, and their translational regulatory properties were analyzed by measuring the repression of the b-galactosidase level in the presence of a plasmid overproducing S15 in trans (see Table 1).