Cationic metals promote sequence-directed DNA bending

Laundon, Caroline H.; Griffith, Jack D.

doi:10.1021/bi00387a003

Cited by 80 publications

(47 citation statements)

References 27 publications

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“…In the case of the mtr and tyrPϩ3* promoters, the HU-or IHF-mediated DNA bending could affect the promoter activities by introducing ratelimiting steps for transcription initiation. This idea is supported by our finding that BaCl 2 , a chemical compound which is known to induce DNA bending (22), can also cause inhibition of transcription of both the mtr and tyrPϩ3 promoters (results not shown). In vitro studies by Flashner and Gralla (9) have shown that BaCl 2 has the same inhibitory or stimulatory effects on the binding of a number of E. coli regulatory proteins to their DNA targets as HU.…”

Section: Fig 2 (A)supporting

confidence: 60%

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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Section: Fig 2 (A)supporting

confidence: 60%

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

1996

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“…Changes in DNA conformation can be potentiated by particular cations such as spermidine (15)(16)(17)(18). These polymorphic variations have been detected because ofthe easily measured changes in physical properties associated with the alterations.…”

Section: Methodsmentioning

confidence: 99%

DNA and spermidine provide a switch mechanism to regulate the activity of restriction enzyme Nae I.

Conrad

Topal

1989

Proc. Natl. Acad. Sci. U.S.A.

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Sequence-specific DNA-protein interactions are basic to DNA function. To better understand these interactions, we studied the effect of position on cleavage of DNA by the type II restriction enzyme (EC 3.1.21.4) Nae I. We discovered two classes of Nae I restriction sites: sites susceptible and sites resistant to cleavage. Kinetic analysis showed that Nae I was activated by DNA containing cleavable Nae I sites to rapidly cleave resistant Nae I sites by a noncompetitive mechanism with a Km for substrate DNA of about 2 nM and a KA for activating DNA of about 6 nM; activation increased catalysis but not substrate binding. Deletion mutagenesis in vitro showed that sequences flanking the Nae I recognition site were responsible for the differences between activating and nonactivating Nae I sites. The polyamine spermidine had a dramatic effect on the interaction of Nae I with DNA; in the presence of 1 mM spermidine, resistant sites were cleaved rapidly and cleavable DNA inhibited cleavage. The direct regulation of enzymatic activity by DNA sequences in trans, and the modulation of this regulation by a polyamine that is sensitive to the cell cycle, provides a regulatory switch mechanism. The implications of this switch for biological control functions are discussed.The type II restriction/modification systems (1), like the repressors (2), are important model systems for sequencespecific DNA-protein interactions that are basic to many biological processes. In the course of studying these interactions, we made the quite unexpected discovery that cleavage by the type II restriction enzyme Nae I can be controlled by specific DNA sequences; the ability to cleave and the action of external DNA sequences were in turn dramatically affected by spermidine. Nae I is isolated from Nocardia aerocolonigenes, a member of a common family, Nocardiaceae, of soil actinomycetes. It cleaves within the sequence GCC/GGC (ref. 3 and supplier catalogs) and, like other type II enzymes, requires only Mg2+ for activity and exhibits only a single function, cleavage. Using commercially available Nae I, we found DNA sequences containing Nae I recognition sites that were cleaved rapidly and Nae I sites that were almost totally resistant to cleavage. Studies of Nae I interaction with these sites showed that Nae I was activated to rapidly cleave resistant sites and that the activator was a cleavable Nae I site. The activated cleavage reaction followed MichaelisMenten kinetics, which indicated that activation was noncompetitive and worked by increasing catalysis rather than by increasing the affinity of the enzyme for its substrate. Deletion mutagenesis in vitro showed that sequences flanking the Nae I recognition site were responsible for the enzymeregulatory properties of the cleavable sites. Spermidine could reverse the effect; in the presence of 1 mM spermidine, resistant sites were cleaved rapidly but cleavable DNA inhibited their cleavage, providing a regulatory switch mechanism. MATERIALS AND METHODSRestriction enzyme cleavage reactions w...

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“…In vitro transcriptional analysis using a supercoiled template has shown that transcription from the mtr promoter ( 70 ) is strongly inhibited by histone-like proteins HU and IHF or by BaCl 2 (26). In the presence of any of these reagents, which are known to alter DNA structure (6,13), it is possible to demonstrate activation of transcription by purified TyrR protein and either of the cofactors, phenylalanine and tryptophan (26). The transcription activation of mtr does not occur if the activation-negative TyrR protein RQ10 is substituted for the wild-type protein or if RNA polymerase with a truncated ␣ C-terminal domain is used (26).…”

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confidence: 99%

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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Cationic metals promote sequence-directed DNA bending

Cited by 80 publications

References 27 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

DNA and spermidine provide a switch mechanism to regulate the activity of restriction enzyme Nae I.

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

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