Trap-mediated transcription termination within the b. Subtilis trp operon leader region
Merlino, Natalie Maria
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Transcription of the tryptophan operon in Bacillus subtilis is regulated by an attenuation mechanism involving competing RNA secondary structures. trp RNA-binding attenuation protein (TRAP) binding to a site within the leader region induces transcription termination. Initially TRAP binding was thought to cause termination by changing the secondary structure of the leader region RNA so as to allow an intrinsic terminator ( trp attenuator) to form. However, the trp attenuator does not function as an intrinsic terminator in the absence of TRAP. Therefore, I further examined the role of the RNA in TRAP-mediated transcription termination. This study shows that TRAP is able to cause efficient transcription termination in vivo when the trp attenuator is mutated or completely removed. However, these mutations in the trp attenuator prevent TRAP from causing transcription termination in vitro. We find that NusA, timing of TRAP binding, and an as yet uncharacterized additional protein(s) from B. subtilis all play a role in the differences observed between the in vitro and in vivo regulation by TRAP. The ability of TRAP to induce transcription termination at mutant attenuators suggests that TRAP has an additional role beyond altering the RNA secondary structure. This additional role appears to involve causing forward translocation of RNA polymerase (RNAP) to induce transcription termination, possibly by specifically interacting with RNAP. I performed a genetic selection to find TRAP mutants that were unable to induce efficient transcription termination (possibly due to a defect in interacting with RNAP). A substitution in TRAP at Glu60 to Lys decreased the efficiency of TRAP-mediated transcription termination but did not alter its RNA or tryptophan affinity. This residue is also required to stabilize an interaction between TRAP and RNAP. Therefore, an acidic amino acid at residue 60 is important for complete transcription termination of B. subtilis RNAP within the trp leader region.