Tryptophan-induced activation of TRAP in Bacillus subtilis
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The tryptophan biosynthetic operon in Bacillus subtilis and other related bacilli is negatively regulated by an RNA binding protein called TRAP ( trp RNA-binding attenuation protein). TRAP is composed of eleven identical subunits arranged in a symmetric ring. Upon binding tryptophan TRAP is activated to bind RNA. Four TRAP residues including, Glu36, Lys37, Lys56 and Arg58, interact directly with the bound RNA. In comparison to the TRAP-RNA interaction' relatively little is known about how tryptophan induces TRAP. Each TRAP bound tryptophan makes nine hydrogen bonds with eight TRAP residues. Of these, Thr30, which is essential for TRAP activation, is especially interesting as substituting Val for Thr at this position results in a TRAP mutant that can bind RNA constitutively. Characterization of T30V and T30VD39A TRAP demonstrated that changes in the vicinity of residue 31 brought about by tryptophan binding are not essential for TRAP activation. Of the four residues involved in interacting with RNA, Glu36, Lys56 and Arg58 are important for RNA binding both in WT and T30V TRAP. Lys37 which is important in WT TRAP for RNA binding is dispensable in T30V TRAP. Mutagenesis studies showed that interaction between G1 of GAGUU repeat of RNA and Lys37 & Asp39 is different in WT and T30V TRAP, thereby providing a possible explanation about the reduced RNA affinity of T30V TRAP as compared to WT TRAP. Additionally, changes in any of the RNA binding residues affect the tryptophan binding properties of the TRAP protein. These observations combined with earlier observations that changes in tryptophan binding residues affects RNA binding of the protein imply a two way exchange of information between the tryptophan and RNA binding sites. A genetic selection scheme for isolation of TRAP mutants capable of binding RNA constitutively was developed. The scheme utilizes the natural biosynthetic pathway of B. subtilis and consists of selection using 5 fluoroindole (5FI), an analog of tryptophan. Approximately 100,000 colonies were tested for survival on 5FI with no positive results. The procedure appears to be too stringent and requires modifications as a TRAP mutant with K d [approximate] 100nM did not survive the selection.