Translation Control of trpG from Transcripts Originating from the Folate Operon Promoter of Bacillus subtilis Is Influenced by Translation-Mediated Displacement of Bound TRAP, While Translation Control of Transcripts Originating from a Newly Identified trpG Promoter Is Not

Abstract

Bacillus subtilis trpG encodes a glutamine amidotransferase subunit that participates in the biosynthesis of both tryptophan and folic acid. TRAP inhibits translation of trpG in response to tryptophan by binding to a site that overlaps the trpG Shine-Dalgarno sequence, thereby blocking ribosome binding. Similar mechanisms regulate trpP and ycbK translation. The equilibrium binding constants of tryptophan-activated TRAP for the trpG, ycbK, and trpP transcripts were determined to be 8, 3, and 50 nM, respectively. Despite TRAP having a higher affinity for the trpG transcript, TRAP exhibited the least control of trpG expression. The trpG ShineDalgarno sequence overlaps the stop codon of the upstream pabB gene, while six of nine triplet repeats within the TRAP binding site are located upstream of the pabB stop codon. Thus, ribosomes translating the upstream pabB cistron could be capable of reducing TRAP-dependent control of TrpG synthesis by displacing bound TRAP. Expression studies using pabB-trpG -lacZ fusions in the presence or absence of an engineered stop codon within pabB suggest that translation-mediated displacement of bound TRAP reduces TRAP-dependent inhibition of TrpG synthesis from transcripts originating from the folate operon promoter (P pabB ). A new trpG promoter (P trpG ) was identified in the pabB coding sequence that makes a larger contribution to trpG expression than does P pabB . We found that TRAP-dependent regulation of trpG expression is more extensive for a transcript originating from P trpG and that transcripts originating from P trpG are not subject to translationmediated displacement of bound TRAP.

RNA-based regulation of genes of tryptophan synthesis and degradation, in bacteria

“…This displacement reduces TRAP inhibition of trpG/pabA mRNA translation of these transcripts ( Fig. 10; Yakhnin et al 2007). On transcripts initiated at the internal promoter, p 2 , TRAP can bind and remain at the trpG Shine-Dalgarno sequence, effectively blocking trpG/pabA translation (Yakhnin et al 2007).…”

“…Tryptophan-activated TRAP regulates translation of trpG/pabA, but principally when the transcript is produced from the p 2 promoter ( Fig. 10; Yakhnin et al 2007). Transcripts initiated at the upstream promoter, p 1 , preceding pabB, allow ribosomes translating pabB to displace TRAP bound at the series of XAG repeats in the trpG/pabA Shine-Dalgarno region.…”

“…One promoter, p 1 , precedes pabB, the gene located before trpG/pabA in the folate operon; the second promoter, p 2 , immediately precedes trpG/pabA ( Fig. 10; Yakhnin et al 2007). Tryptophan-activated TRAP regulates translation of trpG/pabA, but principally when the transcript is produced from the p 2 promoter ( Fig.…”

“…However its regulation is principally translational ( Fig. 10; Babitzke et al 1994;Yakhnin et al 2007). A series of XAG repeats overlap the Shine-Dalgarno sequence for trpG/pabA.…”

“…This gene specifies a glutamine amidotransferase polypeptide that serves as a component of two enzyme complexes, one catalyzing o-aminobenzoate synthesis from chorismate, in tryptophan formation, and the second catalyzing p-aminobenzoate synthesis from chorismate, in folate biosynthesis-hence the designation trpG/pabA (Yakhnin et al 2007). As will be described, recent studies have shown that trpG/pabA of the folate operon is transcribed from two promoters, and that synthesis of the TrpG/PabA polypeptide is translationally regulated by the action of tryptophan-activated TRAP (Yakhnin et al 2007). …”