A 5′ RNA stem-loop participates in the transcription attenuation mechanism that controls expression of the Bacillus subtilis trpEDCFBA operon

The trp RNA-binding attenuation protein (TRAP) regulates expression of the Bacillus subtilis trpEDCFBA operon by transcription attenuation. Tryptophan-activated TRAP binds to the nascent trp leader transcript by interacting with 11 (G/U)AG repeats. TRAP binding prevents formation of an antiterminator structure, thereby promoting formation of an overlapping terminator, and hence transcription is terminated before RNA polymerase can reach the trp structural genes;In addition to the antiterminator and terminator, a stem-loop structure is predicted to form at the 5' end of the trp leader transcript. Deletion of this structure resulted in a dramatic increase in expression of a trpE'-'lacZ translational fusion and a reduced ability to regulate expression in response to tryptophan, By introducing a series of point mutations in the 5' stem-loop, we found that both the sequence and the structure of the hairpin are important for its regulatory function and that compensatory changes that restored base pairing partially restored wild-type-like expression levels. Our results indicate that the 5' stem-loop functions primarily through the TRAP-dependent regulatory pathway. Gel shia results demonstrate that the 5' stem-loop increases the affinity of TRAP for trp leader RNA four- to fivefold, suggesting that the 5' structure interacts with TRAP. In vitro transcription results indicate that this 5' structure functions in the attenuation mechanism, since deletion of the stem-loop caused an increase in transcription readthrough. An oligonucleotide complementary to a segment of the 5' stem-loop was used to demonstrate that formation of the 5' structure is required for proper attenuation control of this operon.