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

Abstract

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.

Kinetic Basis of the Bifunctionality of SsoII DNA Methyltransferase

“…The general scheme of transcription regulation in the SsoII-like RM systems has been described elsewhere [ 84 , 85 ]. The details of this model on the basis of our data are presented in Figure 11 ( Supplementary Information ).…”

“…Because the regulatory site is located closer to the MTase promoter than to the REase promoter, the steric competition between M.SsoII and RNAP for the binding site was suggested [ 85 ]. Our results on bending of the regulatory site by M.SsoII and the DNA−protein complex isomerisation support this notion.…”

Flexibility of the Linker between the Domains of DNA Methyltransferase SsoII Revealed by Small-Angle X-Ray Scattering: Implications for Transcription Regulation in SsoII Restriction–Modification System

“…This ability is based on M.SsoII binding to the regulatory site in the promoter region of the SsoII R–M system [12] . M.SsoII forms a stable complex with the regulatory site, which competes with RNA polymerase and therefore prevents transcription of ssoIIM gene [14] , [55] . This effect decreases the concentration of M.SsoII in the cell thus forming a regulatory circuit with a negative feedback.…”

“…This effect decreases the concentration of M.SsoII in the cell thus forming a regulatory circuit with a negative feedback. The ssoIIR gene promoter is weaker than the ssoIIM gene promoter and therefore repression of ssoIIM transcription stimulates ssoIIR gene transcription indirectly [14] , [55] . Thus, the regulatory activity of M.SsoII is in anticorrelation with its main function, DNA methylation.…”