Expression of the trpEDCFBA operon is regulated at both the transcriptional and translational levels by the trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis. When cells contain sufficient levels of tryptophan to activate TRAP, the protein binds to trp operon transcripts as they are being synthesized, most often causing transcription termination. However, termination is never 100% efficient, and transcripts that escape termination are subject to translational control. We determined that TRAP-mediated translational control of trpE can occur via a novel RNA conformational switch mechanism. When TRAP binds to the 5-untranslated leader segment of a trp operon read-through transcript, it can disrupt a large secondary structure containing a portion of the TRAP binding target. This promotes refolding of the RNA such that the trpE Shine-Dalgarno sequence, located more than 100 nucleotides downstream from the TRAP binding site, becomes sequestered in a stable RNA hairpin. Results from cell-free translation, ribosome toeprint, and RNA structure mapping experiments demonstrate that formation of this structure reduces TrpE synthesis by blocking ribosome access to the trpE ribosome binding site. The role of the Shine-Dalgarno blocking hairpin in controlling translation of trpE was confirmed by examining the effect of multiple nucleotide substitutions that abolish the structure without altering the Shine-Dalgarno sequence itself. The possibility of protein-mediated RNA refolding as a general mechanism in controlling gene expression is discussed.Studies on the regulation of protein synthesis have shown that the RNA secondary structural features present in the 5Ј-UTR 1 dramatically influence translation initiation in both prokaryotic and eukaryotic organisms (for recent reviews see Refs. 1-7). In prokaryotic mRNAs, a conserved stretch of 4 -6 nucleotides called the Shine-Dalgarno (SD) sequence is usually found 4 -11 nucleotides upstream of the initiation codon. The SD sequence base pairs with the 16 S rRNA present in the 30 S ribosomal subunit and thereby correctly positions the initiation codon in the ribosome (8, 9). Translational control mechanisms have been identified in prokaryotes that involve blocking the SD sequence either by RNA secondary structure (9 -12) or by a bound protein (13-16). In the known translational control mechanisms that occur by formation of SD blocking hairpins, formation of the inhibitory structure is spontaneous and does not require protein factors.Expression of the Bacillus subtilis tryptophan biosynthetic genes is regulated in response to changes in the intracellular level of tryptophan at both the transcriptional and translational levels (for a recent review, see Ref. 17). Six of the seven trp genes are clustered in the trpEDCFBA operon. Transcription of the trp operon is regulated by an attenuation mechanism in which tryptophan-activated trp RNA-binding attenuation protein (TRAP) binds to 11 closely spaced (G/U)AG repeats (seven GAG and four UAG) (18 -25). TRAP binding to the 11 tr...