Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P51532 (transcriptional activator)
 6,546 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previously, we isolated and characterized six Bacillus subtilis ada mutants that were hypersensitive to methylnitroso compounds and deficient in the adaptive response to alkylation. Cloning of the DNA complementing the defects revealed the presence of an ada operon consisting of two tandem and partially overlapping genes, adaA and adaB. The two genes encoded proteins with methylphosphotriester-DNA methyltransferase and O6-methylguanine-DNA methyltransferase activities, respectively. To locate the six mutations, the ada operon was divided into five overlapping regions of about 350 bp. The fragments of each region were amplified by polymerase chain reaction and analyzed by gel electrophoresis to detect single-strand conformation polymorphism. Nucleotide sequences of the fragments exhibiting mobility shifts were determined. Three of the mutants carried sequence alterations in the adaA gene: the adaA1 and adaA2 mutants had a one-base deletion and insertion, respectively, and the adaA5 mutant had a substitution of two consecutive bases causing changes of two amino acid residues next to the presumptive alkyl-accepting Cys-85 residue. Three mutants carried sequence alterations in the adaB gene: the adaB3 mutant contained a rearrangement, the adaB6 mutant contained a base substitution causing a change of the presumptive alkyl-accepting Cys-141 to Tyr, and the adaB4 mutant contained a base substitution changing Leu-167 to Pro. The adaB mutants produced ada transcripts upon treatment with low doses of alkylating agents, whereas the adaA mutant did not. We conclude that the AdaA protein functions as the transcriptional activator of this operon, while the AdaB protein specializes in repair of alkylated residues in DNA.
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PMID:Molecular analysis of Bacillus subtilis ada mutants deficient in the adaptive response to simple alkylating agents. 174 39

The adaptive response of Escherichia coli protects cells against the mutagenic and toxic effects of alkylating agents. This response is controlled by the Ada protein, which not only functions as the transcriptional activator of the ada and alkA genes but also possesses two DNA methyltransferae activities. Ada is converted into an efficient transcriptional activator by transferring a methyl group from a DNA methylphosphotriester to its own Cys-69 residue and then binds to a DNA sequence (the Ada box) present in both the ada and alkA promoters. Although the Ada protein initially appeared to regulate the ada and alkA genes in a similar fashion, our studies show that the wild-type Ada protein and its truncated derivatives can differentially regulate ada and alkA transcription. In vivo, lower levels of wild-type methylated Ada are needed to activate ada transcription than alkA transcription. In cells exposed to alkylating agents, the N-terminal half of Ada, which contains the DNA-binding domain, is sufficient for efficient activation of alkA, but not ada, transcription. Moreover, truncated derivatives containing 80-90% of Ada are extremely strong constitutive activators of ada but are only inducible activators of alkA transcription. These results suggest that the mechanism by which Ada activates ada transcription differs from that by which it activates alkA transcription.
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PMID:A region of the Ada DNA-repair protein required for the activation of ada transcription is not necessary for activation of alkA. 192 63

By prophage transformation and subcloning, we have obtained Bacillus subtilis DNA fragments that could complement the hypersensitivity of ada (adaptive response deficient) mutants to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The nucleotide sequence contained two open reading frames that were assigned to the genes adaA and adaB, encoding methylphosphotriester-DNA methyltransferase and O6-methylguanine-DNA methyltransferase, respectively. These two genes overlap by 11 bp and comprise a small operon. The 1.6 Kb transcripts derived from the operon were detected in ada+ cells cultured in the presence of MNNG but not in control ada+ cells. From analysis of the syntheses of DNA alkyltransferases in the ada mutant cells harboring the plasmid carrying the complete or partial fragment, we conclude that the adaA gene product functions as a transcriptional activator of the ada operon, while the adaB gene product specializes in repair of mutagenic O6-methylguanine residues. Comparison with Escherichia coli ada operon showed that the two genes correspond to portions of the E. coli ada gene, implicating gene fusion or splitting as the origin of the difference in the organizations of the genes.
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PMID:Bacillus subtilis ada operon encodes two DNA alkyltransferases. 212 Jun 77

The E. coli ada gene positively controls its own expression and that of other genes (alkA, alkB, aidB) involved in repair of DNA alkylation damage. The cloned ada and alkA genes and purified Ada protein have been used in cell-free systems to identify the inducing signal. Self-methylation of the Ada protein by transfer of a methyl group from a phosphotriester in alkylated DNA to a cysteine residue in the protein converts it to an activator of transcription. The covalently modified Ada protein binds specifically to promoter regions containing the sequence d(AAANNAAAGCGCA) immediately upstream of the RNA polymerase binding sites. This is apparently the first example of conversion of a regulatory gene product to a transcriptional activator by a posttranslational modification event.
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PMID:The intracellular signal for induction of resistance to alkylating agents in E. coli. 300 22

Specific cysteine residues at possible methyl acceptor sites of the Ada protein of Escherichia coli were converted to other amino acids by site-directed mutagenesis of the cloned ada gene of E. coli. Ada protein with the cysteine residue at 321 replaced by alanine was capable of accepting the methyl group from the methylphosphotriester but not from O6-methylguanine or O4-methylthymine of alkylated DNA, whereas the protein with alanine at position 69 accepted the methyl group from the methylated bases but not from the methylphosphotriester. These two mutants were used to elucidate the biological significance of repair of the two types of alkylation lesions. Introduction of the ada gene with the Ala69 mutation into an ada- cell rendered the cell more resistant to alkylating agents with respect to both killing and induction of mutations, but the gene with the Ala321 mutation exhibited no such activity. Replacement of the cysteine residue at position 69, but not at position 321, abolished the ability of Ada protein to promote transcription of both ada and alkA genes in vitro. These results are compatible with the idea that methylation of the cysteine residue at position 69 renders Ada protein active as a transcriptional regulator, whilst the cysteine residue at position 321 is responsible for repair of pre-mutagenic and lethal lesions in DNA. The actions of mutant Ada proteins on the ada and alkA promoters in vivo were investigated using an artificially composed gene expression system. When the ada gene with the Ala69 mutation was introduced into the cell, there was little induction of expression of either the ada or the alkA genes, even after treatment with an alkylating agent, in agreement with the data obtained from studies in vitro. With the Ala321 mutation, however, a considerable degree of ada gene expression occurred without adaptive treatment. The latter finding suggests that the cysteine residue at position 321, which is located near the C terminus of the Ada protein, is involved in regulating activity, as the transcriptional activator.
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PMID:Functional sites of the Ada regulatory protein of Escherichia coli. Analysis by amino acid substitutions. 304

The ada gene of Escherichia coli K-12 encodes the regulatory protein for the adaptive response to alkylating agents. A set of plasmids carrying ordered deletions from the 3' end of the ada gene were isolated and characterized. These ada deletions encode fusion proteins that derive their amino termini from ada and their carboxyl termini from the downstream vector sequence that occurs before an in-frame stop codon. Several of these ada deletions encode Ada derivatives that constitutively activate ada transcription to very high levels. A second class of ada deletions encode Ada derivatives that are dominant inhibitors of the inducible transcription of ada but are inducible activators of alkA transcription. In addition, we found that two Ada derivatives containing the same ada sequences but fused to different vector-derived tails have strikingly different properties. One Ada derivative constitutively activates both ada and alkA expression to very high levels. In contrast, the other Ada derivative is an inducible activator of ada expression, like the wild-type Ada protein, but is not an inducible activator of alkA transcription. Our data suggest that the carboxyl terminus of the Ada protein plays a key role in modulating the ability of the Ada protein to function as a transcriptional activator.
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PMID:Alteration of the carboxyl-terminal domain of Ada protein influences its inducibility, specificity, and strength as a transcriptional activator. 314 84

The ada gene of Escherichia coli encodes a 39-kDa protein which serves both as a transcriptional activator of the adaptive response to alkylating agents and as a DNA repair enzyme demethylating O6-methyl-guanine and phosphotriester residues. Here, the isolated Ada protein was found to be readily cleaved into two fragments of similar size by treatment with trypsin, chymotrypsin, subtilisin, or V8 protease. The fragments retained their respective methyltransferase activities. The Ada protein is, therefore, comprised of two stable active domains united by a central hinge region of about 10 amino acids. Post-translational modification of the Ada protein by methylation of a specific cysteine residue in the NH2-terminal domain is known to convert it to an efficient transcriptional activator. This residue has now been identified as Cys-69.
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PMID:Functional domains and methyl acceptor sites of the Escherichia coli ada protein. 316 36

The Escherichia coli aidB gene is part of the adaptive response to DNA methylation damage. Genes belonging to the adaptive response are positively regulated by the ada gene; the Ada protein acts as a transcriptional activator when methylated in one of its cysteine residues at position 69. Through DNaseI protection assays, we show that methylated Ada (meAda) is able to bind a DNA sequence between 40 and 60 base pairs upstream of the aidB transcriptional startpoint. Binding of meAda is necessary to activate transcription of the adaptive response genes; accordingly, in vitro transcription of aidB is dependent on the presence of meAda. Unmethylated Ada protein shows no protection against DNaseI digestion in the aidB promoter region nor does it promote aidB in vitro transcription. The aidB Ada-binding site shows only weak homology to the proposed consensus sequences for Ada-binding sites in E. coli (AAANNAA and AAAGCGCA) but shares a higher degree of similarity with the Ada-binding regions from other bacterial species, such as Salmonella typhimurium and Bacillus subtilis. Based on the comparison of five different Ada-dependent promoter regions, we suggest that a possible recognition sequence for meAda might be AATnnnnnnG-CAA. Higher concentrations of Ada are required for the binding of aidB than for the ada promoter, suggesting lower affinity of the protein for the aidB Ada-binding site. Common features in the Ada-binding regions of ada and aidB are a high A/T content, the presence of an inverted repeat structure, and their position relative to the transcriptional start site. We propose that these elements, in addition to the proposed recognition sequence, are important for binding of the Ada protein.
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PMID:Transcriptional activation of the Escherichia coli adaptive response gene aidB is mediated by binding of methylated Ada protein. Evidence for a new consensus sequence for Ada-binding sites. 771 36

The ada gene of Escherichia coli K-12 encodes the 39-kDa Ada protein, which consists of two domains joined by a hinge region that is sensitive to proteolytic cleavage in vitro. The amino-terminal domain has a DNA methyltransferase activity that repairs the S-diastereoisomer of methylphosphotriesters while the carboxyl-terminal domain has a DNA methyltransferase activity that repairs O6-methylguanine and O4-methylthymine lesions. Transfer of a methyl group to Cys-69 by repair of a methylphosphotriester lesion converts Ada into a transcriptional activator of the ada and alkA genes. Activation of ada, but not alkA, requires elements contained within the carboxyl-terminal domain of Ada. In addition, physiologically relevant concentrations of the unmethylated form of Ada specifically inhibit methylated Ada-promoted ada transcription both in vitro and in vivo and it has been suggested that this phenomenon plays a pivotal role in the down-regulation of the adaptive response. A set of site-directed mutations were generated within the hinge region, changing the lysine residue at position 178 to leucine, valine, glycine, tyrosine, arginine, cysteine, proline, and serine. All eight mutant proteins have deficiencies in their ability to activate ada transcription in the presence or absence of a methylating agent but are proficient in alkA activation. AdaK178P (lysine 178 changed to proline) is completely defective for the transcriptional activation function of ada while it is completely proficient for transcriptional activation of alkA. In addition, AdaK178P possesses both classes of DNA repair activities both in vitro and in vivo. Transcriptional activation of ada does not occur if both the amino- and carboxyl-terminal domains are produced separately within the same cell. The mutation at position 178 might interfere with activation of ada transcription by changing a critical contact with RNA polymerase, by causing a conformational change of Ada, or by interfering with the communication of conformational information between the amino- and the carboxyl-terminal domains. These results indicate that the hinge region of Ada is important for ada but not alkA transcription and further support the notion that the mechanism(s) by which Ada activates ada transcription differs from that by which it activates transcription at alkA.
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PMID:Alteration of lysine 178 in the hinge region of the Escherichia coli ada protein interferes with activation of ada, but not alkA, transcription. 786 1

The adaptive response of Escherichia coli protects the cells against the toxic and mutagenic effects of certain alkylating agents. The major effector molecule regulating this response is the 39-kDa Ada protein, which functions as both a DNA repair protein and a transcriptional activator. Ada removes methyl groups from phosphotriester and O6-methylguanine lesions in DNA, irreversibly transferring them to cysteine residues at positions 69 and 321, respectively. When methylated at Cys-69, Ada is converted into a potent activator of ada and alkA transcription and binds to a sequence (Ada box) present in both promoters. We have found that physiologically relevant higher concentrations of unmethylated Ada are able to inhibit the activation of ada transcription by methylated Ada, both in vitro and in vivo. In contrast, the same concentrations of unmethylated Ada do not inhibit the activation of alkA transcription by methylated Ada, either in vitro or in vivo. Deletion of the carboxyl-terminal 67 amino acids of Ada abolished the ability of the unmethylated form of the protein to inhibit activation of ada transcription but not the ability of the methylated form to activate ada or alkA transcription. Our results suggest that the Ada protein plays a pivotal role in the negative modulation of its own synthesis and therefore in the down-regulation of the adaptive response. Elements present in the carboxyl terminus of Ada appear to be necessary for this negative regulatory function.
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PMID:The Ada protein acts as both a positive and a negative modulator of Escherichia coli's response to methylating agents. 793 81


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