TyrR Regulon Research Articles

Articles of Significant Interest in This Issue.

The development of novel strategies allowed Camakaris et al. (e00252-21) to isolate mutants of RpoA (α-subunit of RNA polymerase) that were defective in activation of genes of the TyrR regulon and to subsequently isolate suppressors in TyrR that partially restored activation. These data demonstrated that activation involves protein-protein interaction and helped to identify both the “TyrR-specific determinant” on RpoA and the complementary “activation patch” on the TyrR N-terminal domain, both of which are surface exposed. These studies suggest a model for activation by TyrR that involves three sites on the α subunit of the C-terminal domain, the “TyrR-specific determinant,” the “265 determinant,” and the “261 determinant.”

Characterization of the TyrR Regulon in the Rhizobacterium Enterobacter ludwigii UW5 Reveals Overlap with the CpxR Envelope Stress Response.

The TyrR transcription factor controls the expression of genes for the uptake and biosynthesis of aromatic amino acids in Escherichia coli In the plant-associated and clinically significant proteobacterium Enterobacter ludwigii UW5, the TyrR orthologue was previously shown to regulate genes that encode enzymes for synthesis of the plant hormone indole-3-acetic acid and for gluconeogenesis, indicating a broader function for the transcription factor. This study aimed to delineate the TyrR regulon of E. ludwigii by comparing the transcriptomes of the wild type and a tyrR deletion strain. In E. ludwigii, TyrR positively or negatively regulates the expression of over 150 genes. TyrR downregulated expression of envelope stress response regulators CpxR and CpxP through interaction with a DNA binding site in the intergenic region between divergently transcribed cpxP and cpxR Repression of cpxP was alleviated by tyrosine. Methyltransferase gene dmpM, which is possibly involved in antibiotic synthesis, was strongly activated in the presence of tyrosine and phenylalanine by TyrR binding to its promoter region. TyrR also regulated expression of genes for aromatic catabolism and anaerobic respiration. Our findings suggest that the E. ludwigii TyrR regulon has diverged from that of E. coli to include genes for survival in the diverse environments that this bacterium inhabits and illustrate the expansion and plasticity of transcription factor regulons.IMPORTANCE Genome-wide RNA sequencing revealed a broader regulatory role for the TyrR transcription factor in the ecologically versatile bacterium Enterobacter ludwigii beyond that of aromatic amino acid synthesis and transport that constitute the role of the TyrR regulon of E. coli In E. ludwigii, a plant symbiont and human gut commensal, the TyrR regulon is expanded to include genes that are beneficial for plant interactions and response to stresses. Identification of the genes regulated by TyrR provides insight into the mechanisms by which the bacterium adapts to its environment.

Regulation of indole-3-acetic acid biosynthesis by branched-chain amino acids in Enterobacter cloacae UW5.

The soil bacterium Enterobacter cloacae UW5 produces the rhizosphere signaling molecule indole-3-acetic acid (IAA) via the indolepyruvate pathway. Expression of indolepyruvate decarboxylase, a key pathway enzyme encoded by ipdC, is upregulated by the transcription factor TyrR in response to aromatic amino acids. Some members of the TyrR regulon may also be controlled by branched-chain amino acids and here we show that expression from the ipdC promoter and production of IAA are downregulated by valine, leucine and isoleucine. Regulation of the IAA synthesis pathway by both aromatic and branched-chain amino acids suggests a broader role for this pathway in bacterial physiology, beyond plant interactions.

The TyrR transcription factor regulates the divergent akr-ipdC operons of Enterobacter cloacae UW5.

The TyrR transcription factor regulates genes involved in the uptake and biosynthesis of aromatic amino acids in Enterobacteriaceae. Genes may be positively or negatively regulated depending on the presence or absence of each aromatic amino acid, all three of which function as cofactors for TyrR. In this report we detail the transcriptional control of two divergently transcribed genes, akr and ipdC, by TyrR, elucidated by promoter fusion expression assays and electrophoretic mobility shift assays to assess protein-DNA interactions. Expression of both genes was shown to be controlled by TyrR via interactions with two TyrR boxes located within the akr-ipdC intergenic region. Expression of ipdC required TyrR bound to the proximal strong box, and is strongly induced by phenylalanine, and to a lesser extent by tryptophan and tyrosine. Down-regulation of akr was reliant on interactions with the weak box, and may also require a second, as yet unidentified protein for further repression. Tyrosine enhanced repression of akr. Electrophoretic mobility shift assays demonstrated that TyrR interacts with both the strong and weak boxes, and that binding of the weak box in vitro requires an intact adjacent strong box. While the strong box shows a high degree of conservation with the TyrR binding site consensus sequence, the weak box has atypical spacing of the two half sites comprising the palindromic arms. Site-directed mutagenesis demonstrated sequence-specific interaction between TyrR and the weak box. This is the first report of TyrR-controlled expression of two divergent protein-coding genes, transcribed from independent promoters. Moreover, the identification of a predicted aldo-keto reductase as a member of the TyrR regulon further extends the function of the TyrR regulon.

The small RNA RybA regulates key-genes in the biosynthesis of aromatic amino acids under peroxide stress in E. coli

In bacteria, adaptive response to external stimuli is often regulated by small RNAs (sRNAs). In Escherichia coli, the organism in which sRNAs have been best characterized so far, no function could be attributed to 40 out of 79 sRNAs. Here we decipher the function of RybA, one of these orphan sRNAs. RybA was discovered in 2001 by Wassarman et al. using comparative genomics. This sRNA is conserved between E. coli, Salmonella typhimurium and Klebsiella pneumoniae. We determined the expression pattern of RybA under different growth conditions and identified its exact 5′ and 3′ ends. Using microarray and Northern analysis we show that, under peroxide stress, the absence of RybA leads to an upregulation of key genes of the TyrR regulon involved in the metabolism of aromatic compounds including the aromatic amino acids. Although containing an open reading frame, which might have an independent function, RybA does not require translation for this activity and therefore acts at the RNA level. Furthermore we demonstrate that regulation requires the transcription regulator TyrR. The mechanism of activation of TyrR, probably the primary target of RybA, remains to be elucidated. The downregulation of aromatic amino acid biosynthesis might regulate the cellular concentration of chorismate and its availability for other downstream products like ubiquinone or enterobactin. While ubiquinone participates in the defense against oxidative stress in the cytoplasmic membrane, enterobactin is involved in iron import and is therefore detrimental under oxidative stress.

Biosynthesis of the Aromatic Amino Acids.

This chapter describes in detail the genes and proteins of Escherichia coli involved in the biosynthesis and transport of the three aromatic amino acids tyrosine, phenylalanine, and tryptophan. It provides a historical perspective on the elaboration of the various reactions of the common pathway converting erythrose-4-phosphate and phosphoenolpyruvate to chorismate and those of the three terminal pathways converting chorismate to phenylalanine, tyrosine, and tryptophan. The regulation of key reactions by feedback inhibition, attenuation, repression, and activation are also discussed. Two regulatory proteins, TrpR (108 amino acids) and TyrR (513 amino acids), play a major role in transcriptional regulation. The TrpR protein functions only as a dimer which, in the presence of tryptophan, represses the expression of trp operon plus four other genes (the TrpR regulon). The TyrR protein, which can function both as a dimer and as a hexamer, regulates the expression of nine genes constituting the TyrR regulon. TyrR can bind each of the three aromatic amino acids and ATP and under their influence can act as a repressor or activator of gene expression. The various domains of this protein involved in binding the aromatic amino acids and ATP, recognizing DNA binding sites, interacting with the alpha subunit of RNA polymerase, and changing from a monomer to a dimer or a hexamer are all described. There is also an analysis of the various strategies which allow TyrR in conjunction with particular amino acids to differentially affect the expression of individual genes of the TyrR regulon.

FolA, a New Member of the TyrR Regulon inEscherichia coliK-12

The folA gene was identified as a new member of the TyrR regulon by genomic SELEX. Binding of TyrR to two sites in folA activated its transcription. Mutations in the N-terminal or central domain of TyrR, the alpha subunit of RNA polymerase, or integration host factor all abolished activation of the folA promoter.

The TyrR regulon

The TyrR protein of Escherichia coli can act both as a repressor and as an activator of transcription. It can interact with each of the three aromatic amino acids, with ATP and, under certain circumstances, with the C-terminal region of the alpha-subunit of RNA polymerase. TyrR protein is a dimer in solution but in the presence of tyrosine and ATP it self-associates to form a hexamer. Whereas TyrR dimers can, in the absence of any aromatic amino acids, bind to certain recognition sequences referred to as 'strong TyrR boxes', hexamers can bind to extended sequences including lower-affinity sites called 'weak TyrR boxes', some of which overlap the promoter. There is no single mechanism for repression, which in some cases involves exclusion of RNA polymerase from the promoter and in others, interference with the ability of bound RNA polymerase to form open complexes or to exit the promoter. When bound to a site upstream of certain promoters, TyrR protein in the presence of phenylalanine, tyrosine or tryptophan can interact with the alpha-subunit of RNA polymerase to activate transcription. In one unusual case, activation of a non-productive promoter is used to repress transcription from a promoter on the opposite strand. Regulation of individual transcription units within the regulon reflects their physiological function and is determined by the position and nature of the recognition sites (TyrR boxes) associated with each of the promoters. The intracellular levels of the various forms of the TyrR protein are also postulated to be of critical importance in determining regulatory outcomes. TyrR protein remains a paradigm for a regulator that is able to interact with multiple cofactors and exert a range of regulatory effects by forming different oligomers on DNA and making contact with other proteins. A recent analysis identifying putative TyrR boxes in the E. coli genome raises the possibility that the TyrR regulon may extend beyond the well-characterized transcription units described in this review.

Analysis of interaction of regulatory protein TyrR with DNA

Publisher Summary This chapter describes the methodology used to characterize the linkage between the binding of ligands to TyrR and the interaction of TyrR with DNA. The experimental strategies, based primarily on the analysis of sedimentation behavior, provide a quantitative model for the role of tyrosine as a low molecular weight effector of the TyrR regulon. TyrR is a DNA-binding protein that regulates several genes involved in aromatic amino acid biosynthesis in Escherichia coli . Regulation occurs via the repression or activation of several unlinked operons that comprise the TyrR regulon. It discusses that repression varies considerably in magnitude between the different operons and usually involves the coeffectors ATP and tyrosine whereas activation is ATP independent and requires any of the three aromatic amino acids. Strong boxes bind TyrR strongly in vitro in the absence of coeffectors whereas weak boxes have less identity with the consensus sequence and do not bind TyrR unless there is an adjacent strong box and both ATP and tyrosine are present. In most operons repressed by tyrosine, overlap of the promoter by the operator is predominantly via the weak box.

The various strategies within the TyrR regulation of Escherichia coli to modulate gene expression.

The TyrR Regulon of Escherichia coli comprises eight transcription units whose expression is modulated by the TyrR protein. This protein, which is normally a homodimer in solution, can self-associate to form a hexamer, bind with high affinity to specific DNA sequences (TyrR boxes) and interact with the alpha subunit of the RNA polymerase. These various reactions are influenced by the abundance of one or more of the aromatic amino acids, tyrosine, phenylalanine or tryptophan and by the specific location and sequence of the TyrR boxes associated with each transcription unit. This review describes how these activities can be combined in different ways to produce a variety of responses to varying levels of the three aromatic amino acids.

Interaction between the Escherichia coli Regulatory protein TyrR and DNA: a fluorescence footprinting study.

The Escherichia coli regulatory protein TyrR controls the expression of eight transcription units that encode proteins involved in the biosynthesis and transport of aromatic amino acids. It is a homodimer of 57 600 subunit molecular weight and has a binding site for ATP and weak ATPase activity. In the presence of ATP, TyrR binds tyrosine, which induces self-association of TyrR from a dimer to a hexamer. This report examines the interaction of TyrR with a 42 bp DNA oligonucleotide containing a centrally located binding site for TyrR (TyrR box). Replacement of a thymidine residue with an aminouridine residue at positions 7, 9, 13, 15, 19, 22, and 26 from one end of the 42mer enables labeling with fluorescein and successive placement of the label along the major groove of the DNA. The fluorescence footprinting of the oligonucleotide was followed using steady-state and time-resolved fluorescence methods. Binding of the TyrR dimer caused significant changes in the fluorescent properties of the labels attached to positions 13, 15, and 26, suggesting the involvement of these bases in the binding of the protein. Except for the position 15 conjugate, binding of the TyrR dimer caused little change in fluorescence intensity. Therefore, fluorescence anisotropy was used to follow the binding equilibrium. The fluorescence of the position 15 conjugate increased 1.6-fold on binding TyrR, suggesting that the fluorophore was in close contact with the protein. For all conjugates, the addition of tyrosine at the end of the titration with TyrR increased the anisotropy markedly, suggesting that the hexameric form of TyrR could bind the oligonucleotide. Two rotational correlation times were found for the labeled conjugates: one reflecting the motion of the probe at its point of attachment to the DNA (220-290 ps), the other reflecting the global tumbling of the labeled oligonucleotide (14-21 ns). On binding TyrR, changes in the correlation times and their associated amplitudes and changes in the range of angular motion of the probe depended on the position of the label. Evidence is presented that the binding of the TyrR hexamer, but not the TyrR dimer, affects regions that flank the binding sequence. The results support the hypothesis that the binding of the TyrR hexamer is responsible for interaction between tandem TyrR boxes in the tyrR regulon.

A genetic analysis of various functions of the TyrR protein of Escherichia coli.

The TyrR protein is involved in both repression and activation of the genes of the TyrR regulon. Correction of an error in a previously published sequence has revealed a Cro-like helix-turn-helix DNA-binding domain near the carboxyl terminus. Site-directed mutagenesis in this region has generated a number of mutants that can no longer repress or activate. Deletions of amino acid residues 5 to 42 produced a protein that could repress but not activate. The central domain of TyrR contains an ATP-binding site and is homologous with the NtrC family of activator proteins. A mutation to site A of the ATP-binding site and other mutations in this region affect tyrosine-mediated repression but do not prevent activation or phenylalanine-mediated repression of aroG.

TyrR protein of Escherichia coli and its role as repressor and activator

The TyrR protein regulates the expression of eight transcriptional units that comprise the TyrR regulon. In all but one case, regulation is by repression, while in two cases activation of expression can occur. Notwithstanding the fact that the TyrR protein contains an ATP-binding domain and a helix-turn-helix DNA-binding domain which are structurally homologous to domains of similar functions in proteins such as NifA, NtrC, DctD and XylR, it differs from them in a number of respects. It is not a part of a two-protein component system and it lacks the amino-terminal domain that is present on NtrC and DctD. It activates transcription from 'E sigma 70, promoters but not from 'E sigma 54, promoters. ATP binding seems to be essential for tyrosine-mediated repression but not for activation. In addition, the activity of the TyrR protein is modulated by the binding of one or more of the aromatic amino acids. The consensus sequence for TyrR-binding sites in DNA, referred to as TyrR boxes, is TGTAAAN6TTTACA. Tyrosine-mediated repression occurs at operators containing a pair of adjacent boxes. These have unequal affinities for the TyrR protein. The box that overlaps the RNA polymerase binding site is only bound by TyrR in the presence of both ATP and tyrosine, and binding appears to involve co-operativity between two TyrR protein dimers. In contrast, activation of expression by TyrR appears to require phenylalanine but not ATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular analysis of the regulatory region of the Escherichia coli K-12 tyrB gene.

The tyrB gene from Escherichia coli K-12 was cloned and sequenced, and the transcriptional start point of tyrB was determined by primer extension. By using a fusion plasmid in which the lacZ structural gene is transcribed from the tyrB promoter, it was shown that the expression of tyrB is controlled at the transcriptional level by the TyrR protein, with tyrosine as corepressor. The fusion plasmid was used to isolate mutants in which the repression of tyrB had been abolished. The tyrB promoter-operator region of these mutants was sequenced, and the tyrB operator was identified. A comparison between the tyrB operator and those of the other genes belonging to the tyrR regulon is presented.

Transcription control of the aroP gene in Escherichia coli K-12: analysis of operator mutants.

The nucleotide sequence of the region containing the promoter-operator for the aroP gene was determined. The start site of aroP transcription was identified by using S1 nuclease mapping and primer extension techniques. Examination of the nucleotide sequence revealed the presence of two "TYR R" boxes which are similar to those identified in the regulatory regions of other genes in the tyrR regulon. Bisulfite-induced aroP operator-constitutive mutants were analyzed, and the base-pair changes responsible for alterations in aroP regulation were located within these boxes.