Expression of the ada gene of Escherichia coli in response to alkylating agents. Identification of transcriptional regulatory elements (original) (raw)
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J Mol Biol, 1988
Ada protein plays a central role in the regulatory synthesis of DNA repair enzymes, following exposure of Escherichia coli to alkylating agents. Methyl groups of alkylated DNA are transferred to Ada protein by its own methyltransferase activity and the methylated Ada protein then acts as a positive regulator to overproduce the ada and related gene products. To elucidate regulatory mechanisms for the expression of the ada gene by its own product, we analyzed the ada promoter region by random and site-directed mutagenesis. A series of deletion analyses revealed that a sequence up to 53 nucleotides upstream from the transcription initiation site is required for the controlled expression of the ada gene. Libraries of base substitution mutants were constructed by synthesizing oligonucleotides corresponding to the ada promoter region in the presence of a small amount of all possible sets of nucleotides. Internal deletion and insertion mutants were also constructed with the use of synthetic oligonucleotides. Using these mutants, the-10 and the-35 boxes of the promoter as well as the ada regulatory sequence were identified, the latter being an eightnucleotide sequence, AAAGCGCA. A six-nucleotide stretch between the regulatory sequence and the-35 box, also affected levels of expression of the gene. When the promoter DNAs derived from wild type or base substitution mutants that showed normal expression in vivo were used as templates for transcription in vitro, the adaspecific RNA was formed in the presence of a methylated form of Ada protein. With the DNAs derived from mutants of defective type as templates, no or relatively small amounts of the RNA were synthesized. Some base substitution mutants showed a constitutive expression of the gene in vivo, but this observation did not reconcile with findings in experiments in vitro.
Expression of the ada gene of Escherichia coli in response to alkylating agents
Journal of Molecular Biology, 1988
Ada protein plays a central role in the regulatory synthesis of DNA repair enzymes, following exposure of Escherichia coli to alkylating agents. Methyl groups of alkylated DNA are transferred to Ada protein by its own methyltransferase activity and the methylated Ada protein then acts as a positive regulator to overproduce the ada and related gene products. To elucidate regulatory mechanisms for the expression of the ada gene by its own product, we analyzed the ada promoter region by random and site-directed mutagenesis. A series of deletion analyses revealed that a sequence up to 53 nucleotides upstream from the transcription initiation site is required for the controlled expression of the ada gene. Libraries of base substitution mutants were constructed by synthesizing oligonucleotides corresponding to the ada promoter region in the presence of a small amount of all possible sets of nucleotides. Internal deletion and insertion mutants were also constructed with the use of synthetic oligonucleotides. Using these mutants, the-10 and the-35 boxes of the promoter as well as the ada regulatory sequence were identified, the latter being an eightnucleotide sequence, AAAGCGCA. A six-nucleotide stretch between the regulatory sequence and the-35 box, also affected levels of expression of the gene. When the promoter DNAs derived from wild type or base substitution mutants that showed normal expression in vivo were used as templates for transcription in vitro, the adaspecific RNA was formed in the presence of a methylated form of Ada protein. With the DNAs derived from mutants of defective type as templates, no or relatively small amounts of the RNA were synthesized. Some base substitution mutants showed a constitutive expression of the gene in vivo, but this observation did not reconcile with findings in experiments in vitro.
ESCHERICHIA COLI GENE INDUCTION BY ALKYLATION TREATMENT
1986
Searches for alkylation-inducible ( a i d ) genes of Escherichia coli have been conducted by screening random fusions of the Mu-dl(ApR lac) phage for fusions showing increased &galactosidase activity after treatment with methylating agents, but not after treatments with UV-irradiation. In this report we describe gene fusions that are specifically induced by alkylation treatments. Nine new mutants are described, and their properties are compared with the five mutants described previously. T h e total of 14 fusion mutants map at five distinct genetic loci. They can be further subdivided on the basis of their induction by methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).
Nucleic Acids Research, 1986
The activated Ada protein triggers expression of DNA repair genes in Escherichia coli in response to alkylation damage. Ada also osesses two distinct suicide alkyltransferase activities, for 0-alkylguanines and for alkyl phosphotriesters in DNA. The mutant Ada3 and Ada5 transferases repair 06-methylguanine in DNA 20 and 3000 times more slowly, respectively, than the wild-type Ada protein, but both exhibit normal DNA phosphotriester repair. These same proteins also exhibit delayed and sluggish induction of the ada and alkA genes. Since the C-terminal 06-methylguanine methyltransferase domain of Ada is not implicated in the direct binding of specific DNA sequences, this part of the Ada protein is likely to play an alternative mechanistic role in gene activation, either by promoting Ada dimerization, or via direct contacts with RNA polymerase.
Journal of Bacteriology, 1989
Oligodeoxynucleotide-mediated mutagenesis of the ada gene of Escherichia coli was used to produce two mutant Ada proteins. In mutant I the methyl acceptor Cys-321 for O6-methylguanine was replaced by histidine; and in mutant II the positions of Cys-321 and His-322 of the wild-type protein were inverted. Neither mutant protein had O6-methylguanine-DNA methyltransferase activity, but both retained the phosphotriester-DNA methyltransferase activity involving methyl group transfer to Cys-69. Under the control of the endogenous promoter, synthesis of mutant I protein was undetectable before or after adaptation treatment with promoter, synthesis of mutant I protein was undetectable before or after adaptation treatment with N-methyl-N'-nitro-N-nitrosoguanidine. This appeared to be due to both inhibition of transcription of the mutant gene and degradation of the synthesized protein. On the other hand, mutant II protein was inducible by N-methyl-N'-nitro-N-nitrosoguanidine, although ...
Journal of Biological Chemistry, 1998
DNA base damage products either formed spontaneously or as a result of exposure to various genotoxic agents were examined for their effects on Escherichia coli RNA polymerase-mediated transcription in vitro. Uracil, O 6-methylguanine (O 6-meG), and 8-oxoguanine (8-oxoG) were placed at specific sites downstream from the transcriptional start site on the transcribed strand of a duplex template under the control of the strong tac promoter. In vitro, single-round transcription experiments carried out with purified E. coli RNA polymerase revealed efficient bypass at the three lesions examined and subsequent generation of full-length runoff transcripts. Transcript sequence analysis revealed that E. coli RNA polymerase inserted primarily adenine into the transcript opposite to uracil, uracil opposite to O 6-meG, and either adenine or cytosine opposite to 8-oxoG. Thus, a uracil in the DNA template resulted in a G-to-A transition mutation in the lesion bypass product whereas O 6-meG produced a C-to-U transition mutation and 8-oxoG generated either the correct transcriptional product or a C-to-A transversion mutation. When 8-oxoG was placed within close proximity to the transcriptional start site (within the region required for effective promoter clearance), a reduced of full-length, runoff transcript was observed, indicative of lower promoter clearance. Taken together, these results demonstrate that the DNA base damages studied here may exert significant in vivo effects on gene expression and DNA repair with respect to the production of mutant proteins (transcriptional mutagenesis), or decreased levels of expressed proteins.
Effect of Alkylating Agents on the Expression of Inducible Genes of Escherichia coli
Microbiology, 1986
Increasing doses of alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine, diethyl sulphate and ethylmethane sulphonate cause an inhibition of the expression of the recA and s j A genes of wild-type Escherichia coli. This behaviour was not observed in a lexA.56 mutant which has a defective LexA repressor that is unable to bind to the SOS operator. Furthermore, an ada-1 mutant showed the same behaviour as the wild-type strain indicating that the adaptive proteins are not responsible for the inhibition of recA and s j A at high doses of alkylating agents. These results suggest that the inhibitory effect of these alkylating agents may be found in the interaction between the LexA repressor and the control regions of sfiA and recA. On the other hand, high doses of either UV light or mitomycin C produced only a slight decrease in the induction of recA and s j A , whereas bleomycin had no effect. The fact that a repressor structurally related to LexA repressor, such as Lac1 protein, showed the same behaviour as the LexA repressor when a Lac+ strain was treated with alkylating agents, suggests that these compounds can modify the binding abilities of repressors to DNA, producing a limited or even abolished release of repressors, and so decreasing the expression of inducible genes.
Microbial Biotechnology, 2009
Secondary structures and the short Shine–Dalgarno sequence in the 5′-untranslated region of bacterial mRNAs (UTR) are known to affect gene expression at the level of translation. Here we report the use of random combinatorial DNA sequence libraries to study UTR function, applying the strong, σ32/σ38-dependent, and positively regulated Pm promoter as a model. All mutations in the libraries are located at least 8 bp downstream of the transcriptional start site. The libraries were screened using the ampicillin-resistance gene (bla) as reporter, allowing easy identification of UTR mutants that display high levels of expression (up to 20-fold increase relative to the wild-type at the protein level). Studies of the two UTR mutants identified by a modified screening procedure showed that their expression is stimulated to a similar extent at both the transcript and protein product levels. For one such mutant a model analysis of the transcription kinetics showed significant evidence of a difference in the transcription rate (about 18-fold higher than the wild type), while there was no evidence of a difference in transcript stability. The two UTR sequences also stimulated expression from a constitutive σ70-dependent promoter (P1/Panti-tet), demonstrating that the UTR at the DNA or RNA level has a hitherto unrecognized role in transcription.