Kinetic analysis of TRAP-mediated transcription attenuation in Bacillus sp : The role of the individual (G/U)AG repeats in TRAP binding site
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In Bacillus subtilis and in other related bacilli, transcription of the trpEDCFBA operon is negatively regulated in response to tryptophan by the trp RNA binding protein (TRAP), a protein that contains 11 identical subunits arranged in a ring structure. Tryptophan-activated TRAP binds to the trp leader mRNA by interacting with 11 (G/U)AG repeats and this binding promotes formation of an intrinsic transcription terminator. Since the protein must bind and alter the folding pattern of the RNA before RNA polymerase proceeds past the terminator sequence, the timing of TRAP binding to the nascent RNA is crucial for this regulatory mechanism. Analysis of in vitro transcription complexes showed that TRAP can bind to the nascent RNA before the entire 11 repeat binding site is available; as soon as three or four trinucleotide repeats are available. This initial interaction ensures that, as the rest of the binding site is transcribed, TRAP associates quickly with the mRNA and promotes the changes in the RNA secondary structure. Kinetic studies of TRAP binding to trp leader RNA showed that this interaction follows a two-step conformational change model and further support the data presented above. Investigation of the role of individual trinucleotide repeats in the trp leader RNA for the transcription attenuation mechanism, revealed that the arrangement of the 11 (G/U)AG in the TRAP binding site is critical for this mechanism with respect to both the timing of TRAP binding to trp leader RNA (kinetics) as well as for the necessary changes in the RNA structure induced by the protein interacting with the nascent RNA. Examination of the control of the trpEDCFBA operon both in vivo and in vitro showed that whereas B. halodurans and B. subtilis use similar TRAP proteins to regulate the tryptophan biosynthetic genes, the level of regulation and the strategies used are different.