TRAP, THE TRP RNA-BINDING ATTENUATION PROTEIN OF BACILLUS-SUBTILIS, IS A MULTISUBUNIT COMPLEX THAT APPEARS TO RECOGNIZE G UAG REPEATS IN THE TRPEDCFBA AND TRPG TRANSCRIPTS/

A filter binding assay was developed to study interactions between pur ified TRAP, the trp RNA-binding attenuation protein of Bacillus subtil is, and trp specific transcripts. TRAP formed stable complexes with tr pED-CFBA leader RNA; binding was L-tryptophan-dependent and was comple te within 60 s. TRAP binds to a segment of the trp leader transcript t hat includes part of an RNA antiterminator structure. Binding to this segment allows formation of an RNA terminator structure, thereby promo ting transcription termination. Using several trpEDCFBA leader deletio n transcripts, we identified several closely spaced trinucleotide repe ats (seven GAG and four UAG repeats) in the trp leader transcript that appeared to be required for TRAP binding. We also showed that TRAP bi nds to a segment of the trpG transcript that includes the trpG ribosom e binding site; the nucleotide sequence of this segment contains sever al appropriately spaced trinucleotide repeats (seven GAG, one UAG, and one AAG). TRAP binding to the trpG transcript would block translation initiation. RNA footprint analysis confirmed interaction between TRAP and the trinucleotide repeats in the various transcripts. TRAP, in th e presence or absence of L-tryptophan, appears to consist of 11 or 12 identical 8-kDa subunits. Our findings suggest that each tryptophan-ac tivated TRAP subunit can bind one G/UAG repeat in a target transcript. Multiple protein-RNA interactions are required for stable association .