Identification in a pseudoknot of a U.G motif essential for the regulation of the expression of ribosomal protein S15

Abstract

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 U.G 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 U.G pair by comparing the effects of these mutations on the expression level of S15. The results show that the U.G wobble pair cannot be substituted by A.G, C.A, A.C, G.U, or C.G without loss of the autocontrol. In addition, the base pair C.G, adjacent to the 5' side of U, cannot be flipped or changed to another complementary base pair without also inducing derepression of translation. A unique motif, made of only two adjacent base pairs, U.G/C.G, is essential for S15 autoregulation and is presumably involved in direct recognition by the S15 protein.

Figure 1

Pseudoknot structure of the S15 messenger site and regulatory properties of mutations in stem 1. Base pair changes were introduced in a rpsO–lacZ translational fusion carried in a λ phage. For each mutation, the effect of overexpression of S15 on the level of β-galactosidase was measured and compared with the value of a strain carrying a wild-type fusion (10). The mutated base pairs indicated are described in Table 1. Repression ratios correspond to the ratio of the β-galactosidase units in the control divided by the value in the mutant and are given in the squares. This ratio is 1 when all control is lost and is 22 for the wild type (WT). The recognition determinant, G⋅U/G⋅C, is boxed, and the adjacent base pairs are ovalized. Ribose protection of the messenger binding site against ethylnitrosourea (ENU) in the presence of S15 are taken from ref. and are shown by circles: •, strong protection; ○, weak protection. +1 corresponds to the translational initiation codon.