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 shift 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.