Effects of mutations in the L-tryptophan binding pocket of the trp RNA-binding attenuation protein of Bacillus subtilis

The Bacillus subtilis tryptophan biosynthetic genes are regulated by the tr p RNA-binding attenuation protein (TRAP), Cooperative binding of L-tryptoph an activates TRAP so that it can bind to RNA. The crystal structure reveale d that L-tryptophan forms nine hydrogen bonds with various amino acid resid ues of TRAP, We performed site-directed mutagenesis to determine the import ance of several of these hydrogen bonds in TRAP activation. We tested both alanine substitutions as well as substitutions more closely related to the natural amino acid at appropriate positions. Tryptophan binding mutations w ere identified in vivo having unchanged, reduced, or completely eliminated repression activity. Several of the in vivo defective TRAP mutants exhibite d reduced affinity for tryptophan in vitro but did not interfere with RNA b inding at saturating tryptophan concentrations. However, a 10-fold decrease in TRAP affinity for tryptophan led to an almost complete loss of regulati on, whereas increased TRAP affinity for tryptophan had little or no effect on the in vivo regulatory activity of TRAP. One hydrogen bond was found to be dispensable for TRAP activity, whereas two others appear to be essential for TRAP function, Another mutant protein exhibited tryptophan-independent RNA binding activity. We also found that trp leader RNA increases the affi nity of TRAP for tryptophan.