Three elements are crucial for the programmed frameshifting in translation of dnaX mRNA: a Shine-Dalgarno (SD)-like sequence, a double-shift site, and a 3′ structure. The conformation of the mRNA containing these three elements was investigated using chemical and enzymatic probes. The probing data show that the structure is a specific stem-loop. The bottom half of the stem is more stable than the top half of the stem. The function of the stem-loop was further investigated by mutagenic analysis. Reducing the stability of the bottom half of the stem strongly effects frameshifting levels, whereas similar changes in the top half are not as effective. Stabilizing the top half of the stem gives increased frameshifting beyond the WT efficiency. The identity of the primary RNA sequence in the stem-loop is unimportant, provided that the overall structure is maintained. The calculated stabilities of the variant stem-loop structures correlate with frameshifting efficiency. The SD-interaction and the stem-loop element act independently to increase frameshifting in dnaX.
Structural probing and mutagenic analysis of the stem-loop required for Escherichia coli dnaX ribosomal frameshifting: programmed efficiency of 50%