title: Substitutions of Thr30 provide mechanistic insight into tryptophan-mediated activation of TRAP binding to RNA.
abstract: TRAP is an 11 subunit RNA binding protein that regulates expression of genes involved in tryptophan biosynthesis and transport in Bacillus subtilis. TRAP is activated to bind RNA by binding up to 11 molecules of l-tryptophan in pockets formed by adjacent subunits. The precise mechanism by which tryptophan binding activates TRAP is not known. Thr30 is in the tryptophan binding pocket. A TRAP mutant in which Thr30 is substituted with Val (T30V) does not bind tryptophan but binds RNA constitutively, suggesting that Thr30 plays a key role in the activation mechanism. We have examined the effects of other substitutions of Thr30. TRAP proteins with small beta-branched aliphatic side chains at residue 30 bind RNA constitutively, whereas those with a small polar side chain show tryptophan-dependent RNA binding. Several mutant proteins exhibited constitutive RNA binding that was enhanced by tryptophan. Although the tryptophan and RNA binding sites on TRAP are distinct and are separated by approximately 7.5 A, several substitutions of residues that interact with the bound RNA restored tryptophan binding to T30V TRAP. These observations support the hypothesis that conformational changes in TRAP relay information between the tryptophan and RNA binding sites of the protein.
authors.affiliation: Department of Biological Sciences, University of Buffalo, The State University of New York, Buffalo, NY 14260, USA.
authors.forename: Vandana
authors.initials: V
authors.lastname: Payal
authors.forename: Paul
authors.initials: P
authors.lastname: Gollnick
mesh_terms: D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D015964:Gene Expression Regulation, Bacterial; D009876:Operon; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013912:Threonine / Q000737:chemistry* / Q000235:genetics; D014357:Trypsin / Q000378:metabolism; D014364:Tryptophan / Q000096:biosynthesis / Q000378:metabolism*; D014633:Valine / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D016601:RNA-Binding Proteins; D013912:Threonine; D012313:RNA; D014364:Tryptophan; D014357:Trypsin; D014633:Valine