Further genetic analysis of the activation function of the TyrR regulatory protein of Escherichia coli

Yang, Jiyeon; Camakaris, H; Pittard, A. J.

doi:10.1128/jb.178.4.1120-1125.1996

Cited by 22 publications

(25 citation statements)

References 23 publications

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“…Mutational studies have identified a number of amino acid residues in the N-terminal domain of TyrR which play an essential role in activation (5,33,34). Lawley et al have studied the TyrRmediated activation of the wild-type tyrP promoter by using an in vitro transcription system and have provided experimental evidence indicating that TyrR protein activates the transcription of tyrP through direct contact with the C-terminal portion of the ␣ subunit of RNA polymerase (23).…”

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In vitro transcriptional analysis of TyrR-mediated activation of the mtr and tyrP+3 promoters of Escherichia coli

1996

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In order to understand the mechanism by which the TyrR protein activates transcription from the mtr and tyrP؉3 promoters, we have carried out in vitro transcription experiments with supercoiled DNA templates. We have shown that addition of the histone-like protein HU or integration host factor (IHF) greatly inhibited the transcription from the mtr and tyrP؉3 promoters. In the presence of phenylalanine, the wild-type TyrR protein, but not a mutant TyrR protein (activation negative), was able to relieve the HU-or IHF-mediated inhibition of transcription. In contrast, the alleviation of the HU-or IHF-mediated transcription inhibition by the wild-type TyrR protein did not occur when a mutant RNA polymerase with a C-terminally truncated ␣ subunit was used to carry out the transcription reaction.In Escherichia coli, the expression of both the mtr and tyrP genes, which code for the tryptophan-and the tyrosine-specific permeases, respectively, can be activated at the level of transcription by the TyrR protein. In the case of mtr, the activation by the TyrR protein is mediated either by tyrosine or by phenylalanine. The extent of activation varies from 3-to 10-fold, depending on the genetic background of the strain used (14,26), and increasing the gene dosage of tyrR ϩ by introducing a multicopy plasmid carrying the tyrR ϩ gene into the cell results in the increased activation of the mtr gene (26). In a trpR mutant background, tryptophan can also mediate transcription activation of the mtr gene by TyrR, but the extent of activation is slighter than that mediated by phenylalanine or tyrosine (26). In the case of tyrP, its expression can be activated 2-to 3-fold by TyrR in the presence of phenylalanine or repressed 20-fold by TyrR in the presence of tyrosine (2,20).In vivo and in vitro studies have identified TyrR binding sites (TyrR boxes) in the upstream regions of both the mtr and tyrP genes (2,20,25,26). In mtr, the TyrR box (strong box), which plays a major role in TyrR-mediated activation, is centered at Ϫ76.5 or Ϫ77.5. In tyrP, the TyrR box (strong box), which is responsible for TyrR-mediated activation, is centered at Ϫ64.5. The strong TyrR box of tyrP is not positioned ideally for activation, because moving it 3 to 4 or 13 to 14 bases further upstream increases the extent of activation to 10-fold (1). With the strong box in these positions, both phenylalanine and tyrosine can mediate the activation by TyrR (1).The TyrR polypeptide contains 513 amino acid residues which constitute three structural domains (4, 6, 35). Mutational studies have identified a number of amino acid residues in the N-terminal domain of TyrR which play an essential role in activation (5, 33, 34). Lawley et al. have studied the TyrRmediated activation of the wild-type tyrP promoter by using an in vitro transcription system and have provided experimental evidence indicating that TyrR protein activates the transcription of tyrP through direct contact with the C-terminal portion of the ␣ subunit of RNA polymerase (23). On the basis of these resu...

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“…We also tested the ability of the mutant TyrR protein, TyrR-RQ10, which is defective in activation in vivo (34), to activate transcription in vitro. In this experiment, we showed clearly that TyrR-RQ10 is completely inactive in stimulating transcription from both the mtr and tyrPϩ3* promoters ( Fig.…”

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In vitro transcriptional analysis of TyrR-mediated activation of the mtr and tyrP+3 promoters of Escherichia coli

1996

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“…Alanine-scanning mutagenesis of the tyrR gene has shown that the side chains of arginine-2, aspartate-9, and arginine-10 are most critical for transcription activation of mtr (4,25). It is interesting to note that these three critical amino acids in the TyrR activating region are all charged residues, and out of the eight amino acid residues in ␣CTD whose side chains are involved in TyrR-mediated activation, two (aspartate-258 and glutamate-261) are negatively charged and two (arginine-265 and lysine-297) are positively charged.…”

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“…The TyrR protein contains three structural domains (5), and genetic analysis has mapped the activation function of TyrR to the N-terminal domain (4,24,25). Alanine scanning mutagenesis has identified a number of amino acid residues whose side chains are specifically involved in activation (25).…”

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Amino acid residues in the alpha-subunit C-terminal domain of Escherichia coli RNA polymerase involved in activation of transcription from the mtr promoter

et al. 1997

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To examine the role of the amino acid residues (between positions 258 and 275 and positions 297 and 298) of the ␣-subunit of RNA polymerase in TyrR-mediated activation of the mtr promoter, we have carried out in vitro transcription experiments using a set of mutant RNA polymerases with a supercoiled mtr template. Decreases in factor-independent transcription in vitro by mutant RNA polymerases L262A, R265A, and K297A suggested the presence of a possible UP element associated with the mtr promoter. Mutational studies have revealed that an AT-rich sequence centered at ؊41 of the mtr promoter (SeqA) functions like an UP element. In vivo and in vitro analyses using a mutant mtr promoter carrying a disrupted putative UP element showed that this AT-rich sequence is responsible for interactions with the ␣-subunit which influence transcription in the absence of TyrR protein. However, the putative UP element is not needed for activator-dependent activation of the mtr promoter by TyrR and phenylalanine. The results from in vitro studies indicated that the ␣-subunit residues leucine-262, arginine-265, and lysine-297 are critical for interaction with the putative UP element of the mtr promoter and play major roles in TyrR-dependent transcription activation. The residues at positions 258, 260, 261, 268, and 270 also play important roles in TyrR-dependent activation. Other residues, at positions 259, 263, 264, 266, 269, 271, 273, 275, and 298, appear to play less significant roles or no role in activation of mtr transcription.Activation of transcription initiation plays an important role in modulating gene expression. Recent studies with Escherichia coli have indicated that activation of transcription generally involves direct interactions between RNA polymerase bound at a promoter and a transcriptional activator bound at a site located upstream of the promoter (2,11,18). Based on the results of genetic, structural, and biophysical studies, it has been proposed that the ␣-subunit of RNA polymerase of E. coli is involved in such protein-protein interactions for a number of transcription activators (2, 10-12, 21). Work by Ross et al. (19) showed that the ␣-subunit of RNA polymerase can also interact with an AT-rich sequence (UP element) which is located upstream of the Ϫ35 region of the rrnBp 1 promoter of E. coli and is responsible for enhancement of transcription initiation. Such UP elements appear to be a feature of many promoters (8,19,22).The TyrR protein of E. coli is both a repressor and an activator and controls the expression of a group of eight transcriptional units (TyrR regulon) whose translational products are involved in the biosynthesis or uptake of the three aromatic amino acids (17). The TyrR protein contains three structural domains (5), and genetic analysis has mapped the activation function of TyrR to the N-terminal domain (4,24,25). Alanine scanning mutagenesis has identified a number of amino acid residues whose side chains are specifically involved in activation (25).Transcriptional expression of the mtr...

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“…Critical residues have been identified at positions 9, 10, 93 and 103 (Yang et al 1996a). It has not yet been resolved whether these changes affect ligand binding or directly influence interactions between TyrR and the Q subunit of R N A polymerase.…”

Section: The Amino-terminal Domain and Its Involvement In The Activatmentioning

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

Pittard

1996

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The TyrR Regulon o f 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 a 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.

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Further genetic analysis of the activation function of the TyrR regulatory protein of Escherichia coli

Cited by 22 publications

References 23 publications

In vitro transcriptional analysis of TyrR-mediated activation of the mtr and tyrP+3 promoters of Escherichia coli

In vitro transcriptional analysis of TyrR-mediated activation of the mtr and tyrP+3 promoters of Escherichia coli

Amino acid residues in the alpha-subunit C-terminal domain of Escherichia coli RNA polymerase involved in activation of transcription from the mtr promoter

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

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