SsoII-like DNA-methyltransferase Ecl18kI: Interaction between regulatory and methylating functions

Fedotova, E. A.; Protsenko, A. S.; Zakharova, Marina; Лаврова, Н. В.; Alekseevsky, A.V.; Oretskaya, Tatiana S.; Karyagina, A. S.; Solonin, Alexander S.; Кубарева, Е. А.

doi:10.1134/s0006297909010131

Cited by 13 publications

(14 citation statements)

References 12 publications

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“… 1 Data for M.Ecl18kl, M.SsoII, M.SsoII(C142A), M.Ecl18kl(R15A), M.Ecl18kl(R35A), M.Ecl18kl(R38A), M.Ecl18kl(R39A), and M.Ecl18kl(R42A) have been published earlier [9]. …”

Section: Resultsmentioning

confidence: 99%

Peculiarities of the Regulation of Gene Expression in the Ecl18kI Restriction–Modification System

et al. 2013

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Transcription regulation in bacterial restriction–modification (R–M) systems is an important process, which provides coordinated expression levels of tandem enzymes, DNA methyltransferase (MTase) and restriction endonuclease (RE) protecting cells against penetration of alien DNA. The present study focuses on (cytosine-5)-DNA methyltransferase Ecl18kI (M.Ecl18kI), which is almost identical to DNA methyltransferase SsoII (M.SsoII) in terms of its structure and properties. Each of these enzymes inhibits expression of the intrinsic gene and activates expression of the corresponding RE gene via binding to the regulatory site in the promoter region of these genes. In the present work, complex formation of M.Ecl18kI and RNA polymerase from Escherichia сoli with the promoter regions of the MTase and RE genes is studied. The mechanism of regulation of gene expression in the Ecl18kI R–M system is thoroughly investigated. M.Ecl18kI and RNA polymerase are shown to compete for binding to the promoter region. However, no direct contacts between M.Ecl18kI and RNA polymerase are detected. The properties of M.Ecl18kI and M.SsoII mutants are studied. Amino acid substitutions in the N-terminal region of M.Ecl18kI, which performs the regulatory function, are shown to influence not only M.Ecl18kI capability to interact with the regulatory site and to act as a transcription factor, but also its ability to bind and methylate the substrate DNA. The loss of methylation activity does not prevent MTase from performing its regulatory function and even increases its affinity to the regulatory site. However, the presence of the domain responsible for methylation in the M.Ecl18kI molecule is necessary for M.Ecl18kI to perform its regulatory function.

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“… 1 Data for M.Ecl18kl, M.SsoII, M.SsoII(C142A), M.Ecl18kl(R15A), M.Ecl18kl(R35A), M.Ecl18kl(R38A), M.Ecl18kl(R39A), and M.Ecl18kl(R42A) have been published earlier [9]. …”

Section: Resultsmentioning

confidence: 99%

Peculiarities of the Regulation of Gene Expression in the Ecl18kI Restriction–Modification System

et al. 2013

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“…Their regulation was found to be associated with interference between the two convergent promoters (25,50). In these R–M systems, as well as in MspI (59), two feedback loops have been identified.…”

Section: Discussionmentioning

confidence: 99%

Antisense RNA associated with biological regulation of a restriction–modification system

Mruk

Liu

et al. 2011

Nucleic Acids Research

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Restriction–modification systems consist of a modification enzyme that methylates a specific DNA sequence and a restriction endonuclease that cleaves DNA lacking this epigenetic signature. Their gene expression should be finely regulated because their potential to attack the host bacterial genome needs to be controlled. In the EcoRI system, where the restriction gene is located upstream of the modification gene in the same orientation, we previously identified intragenic reverse promoters affecting gene expression. In the present work, we identified a small (88 nt) antisense RNA (Rna0) transcribed from a reverse promoter (PREV0) at the 3′ end of the restriction gene. Its antisense transcription, as measured by transcriptional gene fusion, appeared to be terminated by the PM1,M2 promoter. PM1,M2 promoter-initiated transcription, in turn, appeared to be inhibited by PREV0. Mutational inactivation of PREV0 increased expression of the restriction gene. The biological significance of this antisense transcription is 2-fold. First, a mutation in PREV0 increased restriction of incoming DNA. Second, the presence of the antisense RNA gene (ecoRIA) in trans alleviated cell killing after loss of the EcoRI plasmid (post-segregational killing). Taken together, these results strongly suggested the involvement of an antisense RNA in the biological regulation of this restriction–modification system.

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“…The operator contains a single rather than a duplicated palindrome and the inverted repeat in the operator is much longer than those in C-boxes. Our recent attempts to observe operator binding with recombinant HTH domain of M.Ecl18kI were unsuccessful (24), indicating that sequences in the methyltrasnferase part of M.Ecl18kI are required for DNA binding (the reverse is not true since M.Ecl18kI lacking the HTH domain is fully capable of site methylation). Structural characterization of M.Ecl18kI, alone and in complex with the operator, will be necessary to clarify these issues.…”

Section: Discussionmentioning

confidence: 99%

Transcription regulation of restriction-modification system Ecl18kI

Bogdanova

Zakharova

Nagornykh³

et al. 2009

Nucleic Acids Research

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Restriction-modification (R-M) system Ecl18kI is representative of R-M systems whose coordinated transcription is achieved through a separate DNA-binding domain of the methyltransferase. M.Ecl18kI recognizes an operator sequence located in the noncoding region that separates the divergently transcribed R and M genes. Here we show that, contrary to previous predictions, the two ecl18kI promoters are not divergent, but actually face one another. The binding of M.Ecl18kI to its operator prevents RNA polymerase (RNAP) binding to the M promoter by steric exclusion, but has no direct effect on RNAP interaction with the R promoter. The start point for R transcription is located outside of the intergenic region, opposite the initiation codon of the M gene. Regulated transcription of the potentially toxic ecl18kI R gene is accomplished (i) at the stage of promoter complex formation, through direct competition from complexes formed at the M promoter, and (ii) at the stage of promoter clearance, since R promoter-bound RNAP escapes the promoter more slowly than RNAP bound to the M promoter.

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SsoII-like DNA-methyltransferase Ecl18kI: Interaction between regulatory and methylating functions

Cited by 13 publications

References 12 publications

Peculiarities of the Regulation of Gene Expression in the Ecl18kI Restriction–Modification System

Peculiarities of the Regulation of Gene Expression in the Ecl18kI Restriction–Modification System

Antisense RNA associated with biological regulation of a restriction–modification system

Transcription regulation of restriction-modification system Ecl18kI

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