Expression of the Bacillus subtilis trpEDCFBA operon is regulated by the interaction of tryptophan-activated TRAP with 11 (G/U)AG trinucleotide repeats that lie in the leader region of the nascent trp transcript. Bound TRAP prevents folding of an antiterminator structure and favors formation of an overlapping intrinsic terminator hairpin upstream of the trp operon structural genes. A 5'-stem-loop (5'SL) structure that forms just upstream of the triplet repeat region increases the affinity of TRAP-trp RNA interaction, thereby increasing the efficiency of transcription termination. Single-stranded nucleotides in the internal loop and in the hairpin loop of the 5'SL are important for TRAP binding. We show here that altering the distance between these two loops suggests that G7, A8, and A9 from the internal loop and A19 and G20 from the hairpin loop constitute two structurally discrete TRAP-binding regions. Photochemical cross-linking experiments also show that the hairpin loop of the 5'SL is in close proximity to the flexible loop region of TRAP during TRAP-5'SL interaction. The dimensions of B. subtilis TRAP and of a three-dimensional model of the 5'SL generated using the MC-Sym and MC-Fold pipeline imply that the 5'SL binds the protein in an orientation where the helical axis of the 5'SL is perpendicular to the plane of TRAP. This interaction not only increases the affinity of TRAP-trp leader RNA interaction, but also orients the downstream triplet repeats for interaction with the 11 KKR motifs that lie on TRAP's perimeter, increasing the likelihood that TRAP will bind in time to promote termination.