TRAP regulates expression of the Bacillus subtilis trpEDCFBA operon by a transcription attenuation mechanism in which tryptophan-activated TRAP binds to 11 (G/U)AG repeats in the nascent trp leader transcript. Bound TRAP blocks formation of an antiterminator structure and allows formation of an overlapping intrinsic terminator upstream of the trp operon structural genes. A 5' stem-loop (5'SL) structure located upstream of the triplet repeat region also interacts with TRAP. TRAP-5'SL RNA interaction participates in the transcription attenuation mechanism by preferentially increasing the affinity of TRAP for the nascent trp leader transcript during the early stages of transcription, when only a few triplet repeats have been synthesized. Footprinting assays indicated that the 5'SL contacts TRAP through two discrete groups of single-stranded nucleotides that lie in the hairpin loop and in an internal loop. Filter binding and in vivo expression assays of 5'SL mutants established that G7, A8, and A9 from the internal loop, and A19 and G20 from the hairpin loop are critical for proper 5'SL function. These nucleotides are conserved among certain other 5'SL-containing organisms. Single-round transcription results indicated that the 5'SL increases the termination efficiency when transcription is fast; however, the influence of the 5'SL was lost when transcription was slowed by reducing the ribonucleoside triphosphate concentration. Since there is a limited amount of time for TRAP to bind to the nascent transcript and promote termination, our data suggest that the contribution of TRAP-5'SL interaction increases the rate of TRAP binding, which, in turn, increases the efficiency of transcription termination.