Physiological Effects of Anti-TRAP Protein Activity and tRNA Trp Charging on trp Operon Expression in Bacillus subtilis

Abstract

ABSTRACT 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 tRNA Trp . In this study, we examined the roles of AT synthesis and tRNA Trp charging in mediating physiological responses to tryptophan starvation. Adding excess phenylalanine to wild-type cultures reduced the charged tRNA 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 tRNA Trp level, implying that phenylalanine, when added alone, reduces the availability of tryptophan for tRNA Trp charging. Changes in the charged tRNA 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 tRNA Trp level. When this shift was performed with an AT-deficient mutant, the charged tRNA 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 tRNA Trp acting as the crucial signal regulating AT production.