Unusual mRNA pseudoknot structure is recognized by a protein translational repressor

Translation of ribosomal proteins in the α operon of E. coli is repressed by one of the encoded proteins, S4; it specifically recognizes an RNA fragment containing the translational initiation site for the first gene in the operon. RNA structure mapping experiements have suggested a pseudoknot structure for the S4 binding site: the loop of a hairpin is base paired to sequences downstream of the hairpin. Here, we systematically test this proposed structure by measuring S4 binding to an extensive set of site-directed mutations that create compensatory base pair changes in potential helices. The pseudoknot folding is confirmed, and two additional, unexpected interactions within the pseudoknot are also detected. The overall structure is an unusual “double pseudoknot” linking a hairpin up-stream of the ribosome binding site with sequences 2–10 codons downstream of the initiation codon. Stabilization of this structure by S4 could account for translational repression.