trp RNA-binding attenuation protein-mediated long distance RNA refolding regulates translation of trpE in Bacillus subtilis

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Abstract

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.

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