trp RNA-binding attenuation protein-5 ' stem-loop RNA interaction is required for proper transcription attenuation control of the Bacillus subtilis trpEDCFBA operon

The trp RNA-binding attenuation protein (TRAP) regulates expression of the Bacillus subtilis trpEDCFBA operon by a novel transcription attenuation mec hanism. Tryptophan-activated TRAP binds to the nascent trp leader transcrip t by interacting with 11 (G/U)AG repeats, 6 of which are present in an anti terminator structure. TRAP binding to these repeats prevents formation of t he antiterminator, thereby promoting formation of an overlapping intrinsic terminator. A third stem-loop structure that forms at the extreme 5' end of the trp leader transcript also plays a role in the transcription attenuati on mechanism. The 5' stem-loop increases the affinity of TRAP for trp leade r RNA. Results from RNA structure mapping experiments demonstrate that the 5' stem-loop consists of a 3-bp lower stem, a 5-by-2 asymmetric internal lo op, a 6-bp upper stem, and a hexaloop at the apex of the structure. Footpri nting results indicate that TRAP interacts with the 5' stem-loop and that t his interaction differs depending on the number of downstream (G/U)AG repea ts present in the transcript. Expression studies with trpE'-'lacZ translati onal fusions demonstrate that TRAP-5' stem-loop interaction is required for proper regulation of the trp operon. 3' RNA boundary experiments indicate that the 5' structure reduces the number of (G/U)AG repeats required for st able TRAP-trp leader RNA association. Thus, TRAP-5' stem-loop interaction m ay increase the likelihood that TRAP will bind to the (G/U)AG repeats in ti me to block antiterminator formation.