Using hetero-11-mers composed of wild type and mutant subunits to study tryptophan binding to TRAP and its role in activating RNA binding.
Expression of genes involved in tryptophan metabolism in Bacillus subtilis is regulated by the TRAP protein in response to changes in l-tryptophan levels. TRAP binding to several RNA targets that contain between 9 and 11 (G/U)AG repeats regulates transcription and/or translation of these genes. TRAP consists of 11 identical subunits and is activated to bind RNA by binding up to 11 molecules of tryptophan. To investigate the mechanism by which tryptophan binding activates TRAP, we generated hetero-11-mers containing different proportions of subunits from wild type (WT) TRAP that bind tryptophan and from a mutant TRAP (Thr(25) to Ala) defective in tryptophan binding. Studies of these hetero-11-mers show that tryptophan-binding sites created from active subunits bind tryptophan with similar affinity to those in WT homo-11-mers, whereas sites containing the T25A substitution do not bind tryptophan. Hetero-11-mers with very few (one or two) bound tryptophans show only 10-fold lower affinity than WT TRAP for an RNA with 11 GAG repeats, whereas TRAP with no bound tryptophan shows no detectable binding to this RNA. We also demonstrate that tryptophan binding induces a conformational change in TRAP in the vicinity of the RNA-binding site, suggesting a possible mechanism for activation of RNA binding.
Li
Pan T X
PT
Department of Biological Sciences, State University of New York, Buffalo, New York 14260, USA.
Gollnick
Paul
P
D001412:Bacillus subtilis / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001704:Biopolymers; D008958:Models, Molecular; D011485:Protein Binding; D012329:RNA, Bacterial / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism*
D001426:Bacterial Proteins; D001704:Biopolymers; C079092:MtrB protein, Bacteria; D012329:RNA, Bacterial; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D014364:Tryptophan