Physiological effects of anti-TRAP protein activity and tRN[A.sup.Trp] charging on trp operon expression in Bacillus subtilis

The Bacillus subtilis anti-TRAP protein regulates the ability of the tryptophan-activated TRAP protein to bind to trp operon leader RNA and promote transcription termination. AT synthesis is regulated both transcriptionally and translationally by uncharged tRN[A.sup.Trp]. In this study, we examined the roles of AT synthesis and tRN[A.sup.Trp] charging in mediating physiological responses to tryptophan starvation. Adding excess phenylalanine to wild-type cultures reduced the charged tRN[A.sup.Trp] level from 80% to 40%; the charged level decreased further, to 25%, in an AT-deficient mutant. Adding tryptophan with phenylalanine increased the charged tRN[A.sup.Trp] level, implying that phenylalanine, when added alone, reduces the availability of tryptophan for tRN[A.sup.Trp] charging. Changes in the charged tRN[A.sup.Trp] level observed during growth with added phenylalanine were associated with increased transcription of the genes of tryptophan metabolism. Nutritional shift experiments, from a medium containing tryptophan to a medium with phenylalanine and tyrosine, showed that wild-type cultures gradually reduced their charged tRN[A.sup.Trp] level. When this shift was performed with an AT-deficient mutant, the charged tRN[A.sup.Trp] level decreased even further. Growth rates for wild-type and mutant strains deficient in AT or TRAP or that overproduce AT were compared in various media. A lack of TRAP or overproduction of AT resulted in phenylalanine being required for growth. These findings reveal the importance of AT in maintaining a balance between the synthesis of tryptophan versus the synthesis of phenylalanine, with the level of charged tRN[A.sup.Trp] acting as the crucial signal regulating AT production.