Expression of the ada gene of Escherichia coli in response to alkylating agents. Identification of transcriptional regulatory elements

Takanori Nakamura, Yasuhito Tokumoto, Kunihiko Sakumi, George Koike, Yusaku Nakabeppu, Mutsuo Sekiguchi

研究成果: Contribution to journalArticle査読

25 被引用数 (Scopus)

抄録

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 eight-nucleotide 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 ada-specific 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.

本文言語英語
ページ(範囲)483-494
ページ数12
ジャーナルJournal of Molecular Biology
202
3
DOI
出版ステータス出版済み - 8 5 1988

All Science Journal Classification (ASJC) codes

  • 構造生物学
  • 分子生物学

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