Evidence for allosteric coupling between the ribosome and repressor binding sites of a translationally regulated mRNA.

Escherichia coli ribosomal protein S4 is a translational repressor regulating the expression of four ribosomal genes in the alpha operon. In vitro studies have shown that the protein specifically recognizes an unusual mRNA pseudoknot secondary structure which links sequences upstream and downstream of the ribosome binding site for rpsM (S13) [Tang, C. K., & Draper, D. E. (1989) Cell 57, 531]. We have prepared fusions of the rpsM translational initiation site and lacZ that allows us to detect repression in cells in which overproduction of S4 repressor can be induced. Twenty-five mRNA sequence variants have been introduced into the S13-lacZ fusions and the levels of translational repression measured. Sets of compensating base changes confirm the importance of the pseudoknot secondary structure for translational repression. An A residue in a looped, single-stranded sequence is also required for S4 recognition and may contact S4 directly. Comparison of translational repression levels and S4 binding constants for the set of mRNA mutations show that nine mutants are repressed much more weakly than predicted from their affinity for S4; in extreme cases no repression can be detected for variants with unchanged S4 binding. We suggest that the mRNA contains functionally distinct ribosome and repressor binding sites that are allosterically coupled. Mutations can relieve translational repression by disrupting the linkage between the two sites without altering S4 binding. This proposal assigns to the mRNA a more active role in mediating translational repression than found in other translational repression systems.

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