trp operon organization, and associated regulatory factors and elements, are “painted” on a phylogenetic tree of organisms. Only trp genes that function for primary tryptophan biosynthesis, not trp genes that participate in specialized secondary pathways, are shown. The branches for Helicobacter pylori and Corynebacterium glutamicum are color coded to indicate the LGT origins of their whole-pathway trp operons. A multiple alignment of the 16S rRNA sequences of representative genomes was used to estimate “genetic similarity distances” using the PROTDIST program of the phylogeny inference package program PHYLIP. Based on these estimated distances, successive clustering of lineages was performed using the neighbor-joining algorithm as implemented in the NEIGHBOR program. The regulatory DNA/RNA binding sites, transcription attenuators, and leader peptides indicated have been identified experimentally or predicted from computer analysis of genome sequences. For large operons with more than 5 consecutive non-trp genes (white arrows), the number of these genes is indicated. Organisms with a tRNA^Trp-regulated T box element controlling transcription of their trp operon generally also contain a tRNA^Trp-regulated T box element controlling transcription of trpS, the structural gene for tryptophanyl-tRNA synthetase. mtrB, rtpA, and ltbR, are, respectively, the structural genes for the TRAP, AT, and LtrB polypeptides.

The variety of factors and elements participating in regulation of transcription of trp biosynthetic operons are shown. Some block the action of RNA polymerase, while others influence premature termination of transcription. The signals that are most often sensed are the intracellular concentration of free Trp, and the level of uncharged tRNA^Trp. These activate or regulate formation of different molecular components, including DNA and RNA binding proteins, RNAs, protein binding proteins, and translating ribosomes. In some instances where regulation by transcription termination has been predicted, the associated signal has not been identified. Transcription regulation based on Trp activation of the TrpR repressor (a, b). The TrpR protein is found mainly in Gammaproteobacteria and in some Chlamydiales (Fig. 1). (a) When intracellular levels of Trp are low, TrpR exists in its inactive aporepressor conformation that cannot bind to trp operator DNA. Consequently, RNA polymerase can initiate trp operon transcription. (b) As the intracellular level of Trp rises, it binds to the TrpR aporepressor, altering its conformation. The repressor then binds at its cognate trp operators, overlapping the trp promoter, preventing initiation of trp operon transcription [10]. Ribosome-mediated transcription attenuation (c, d). In some organisms, the intracellular level of charged tRNA^Trp determines whether a translating ribosome will stall at one of two adjacent trpL Trp codons while attempting synthesis of the TrpL leader peptide. (c) Under growth conditions where the charged tRNA^Trp level is low, the translating ribosome stalls at one of the trpL Trp codons. Ribosome stalling favors formation of the antiterminator structure, rather than the terminator structure, hence transcription continues into the operon. (d) When the level of charged tRNA^Trp is high, translation of trpL mRNA is completed and the translating ribosome dissociates. This allows formation of a Rho-independent transcription terminator in the transcript that terminates transcription in the leader region of the operon. Regulation based on the activation of the TrpI protein (e, f). The TrpI protein belongs to the LysR-family of prokaryotic regulatory proteins. Its structural gene, trpI, is adjacent to and transcribed divergently from the trpBA operon that encodes the subunits of the tryptophan synthase enzyme complex. (e) When the intracellular level of Trp is low, InGP, a biosynthetic intermediate and a tryptophan synthase substrate, is overproduced, favoring double binding of TrpI at operator sites in the trpI-trpBA intergenic region. TrpI binding activates transcription of the trpBA operon, either by direct interaction with RNA polymerase or by inducing DNA bending [43]. (f) Under growth condition where there is excess tryptophan, the InGP concentration is not sufficient to induce recognition of TrpI for its low affinity binding site, thus transcription of trpBA is not activated. Regulation of trp gene expression by TrpI appears to be restricted to Pseudomonas aeruginosa, P. putida, and P. syringae (Fig. 1). Regulation based on the action of the TRAP protein (g, h). TRAP is an RNA-binding protein found in some Bacillales, including Bacillus subtilis, and in a few Clostridia (Fig. 1). (g) Whenever Trp is growth limiting, TRAP is inactive. When TRAP cannot bind to RNA, the leader RNA of the trp operon folds to form the energetically favored secondary structure, the antiterminator. This structure prevents formation of a Rho-independent transcription terminator, consequently transcription proceeds into the operon's structural genes. (h) When there is an excess of tryptophan, TRAP is activated, preparing it to bind to the leader RNA segment of the trp operon transcript. This binding disrupts the antiterminator structure, favoring formation of the terminator structure, leading to premature termination of transcription in the leader region of the operon [17]. Regulation based on the action of the anti-TRAP protein AT (i, j). AT is produced in response to the accumulation of uncharged tRNA. (i) Whenever the uncharged tRNA^Trp level is elevated, AT is synthesized and it binds to Trp-activated TRAP, inhibiting TRAP's activity [44]. (j) When the charged tRNA^Trp level is high, AT synthesis is prevented, eliminating its effect on TRAP function [21,22]. Regulation based on a T box element (k, l). (k) Whenever the charged tRNA^Trp level is low, uncharged tRNA^Trp stabilizes the T box antiterminator structure, preventing formation of the terminator structure. This allows transcription to continue into the remainder of the operon. (l) When the charged tRNA^Trp level is high, this charged tRNA is incapable of stabilizing the antiterminator structure. The terminator therefore forms, terminating transcription [23].