Characterization of the intergenic region which regulates the MspI restriction-modification system

Abstract

The 110-bp intergenic region between mspIM and mspIR, the genes encoding the MspI modification (M.MspI) and restriction (R.MspI) enzymes, respectively, was fused, in both orientations, with lacZ. Expression of a single-copy mspIM-lacZ fusion is more than 400-fold stronger than expression of an mspIR-lacZ fusion. M.MspI in trans represses expression of the mspIM-lacZ fusion by binding to the DNA but does not affect expression of the mspIR-lacZ fusion. Transcription start sites of the genes were identified, and a set of nonoverlapping promoters was assigned. DNase I footprinting showed that M.MspI binds to a site within the intergenic region that includes only the mspIM regulatory elements.Prokaryotic type II restriction-modification (RM) systems consist of a restriction endonuclease (R) and a separate modification methylase (M) encoded by two different open reading frames. The two genes are almost always located close to one another (17), although their orientations vary from system to system. Appropriate regulation of the RM genes is important for the survival of the host. Such regulation has recently been demonstrated for several RM systems, including BamHI (4), PvuII (14), and EcoRII (2, 12, 13).MspI (recognizes 5Ј-CCGG-3Ј) is one of the most widely studied RM systems. It has been cloned (5, 9, 15), and M.MspI has also been overproduced in Escherichia coli (1). Although sequences of about 3.0 kb of DNA containing the mspIM and mspIR genes have been reported (5), the transcriptional start sites have not been determined nor have regulatory elements been defined. Since the two genes separated by an intergenic region of 110 bp are transcribed divergently from complementary strands (5), there could be (i) a common region containing overlapping promoter sequences in opposite orientations, (ii) promoter sequences for one gene within the transcribed region of the other, or (iii) total separation of the two promoters. In a previous report we proposed that expression of the mspIM gene is autoregulated, since M.MspI binds to the DNA containing this intergenic region (12). If the promoter sequences overlapped, such binding might also interfere with the transcription of mspIR.In this study we characterize the properties of the mspI intergenic region by (i) studying its fusions with lacZ in E. coli and the effect of M.MspI in trans on such fusions, (ii) mapping the transcription start sites for both the mspIM and mspIR genes, and (iii) determining the binding sites for the M.MspI protein.Plasmids. A low-copy-number plasmid expressing M.MspI, pACM.MspI, was constructed by cloning the 1,555-bp EcoRISau3A fragment from the multicopy plasmid pM. into the DraI-BamHI site of pACYC177 after end filling the EcoRI site. Plasmid pRS415 (11) is a pBR322 derivative fusion vector which allows expression of ␤-galactosidase (␤-Gal) from operon fusions created by cloning a suitable promoter 5Ј to the promoterless lacZ gene (lacZ ϩ ). Plasmid pBW201 (16) expresses M.BsuFI, an isoschizomer of M.MspI.Fusion of the MspI intergenic region to t...

Restriction-Modification Systems as Minimal Forms of Life

“…For example, EcoRII methyltransferase (M.EcoRII) represses its own expression at the transcriptional level (Som and Friedman 1993). Likewise, M.MspI represses, in trans, the expression of the mspIM-lacZ fusion by binding to the intergenic region between mspIM and mspIR (Som and Friedman 1997). Moreover, in lacZ fusion experiments, M.SsoII was shown to repress its own synthesis but to stimulate expression of the cognate restriction endonuclease.…”

A dichotomous epigenetic mechanism governs expression of the LlaJI restriction/modification system

“…M1.LlaJI contains an N‐terminal extension of approximately 85 amino acids, upstream from conserved Motif I (Accession no. ), reminiscent of a number of prokaryotic MTases possessing such a structural feature, which in some cases was shown to contain a DNA binding motif specific to their own promoter, thereby acting as transcriptional repressors (Som and Friedman, 1994; 1997; Karyagina et al ., 1997; Butler and Fitzgerald, 2001). Analysis of the amino acid sequence of this extension on M1.LlaJI revealed the presence of a putative HTH motif (FHLEQLEDFKDLSKSVCSQLKQ) using the Dodd and Egan method, although the score obtained was low and insignificant relative to known HTH motifs identified elsewhere (Dodd and Egan, 1990).…”

“…A number of other Type II systems, such as SsoII (Karyagina et al ., 1997), EcoRII (Som and Friedman, 1994), MspI (Som and Friedman, 1997) and ScrFI (Butler and Fitzgerald, 2001), exert an alternative mode of transcriptional control. For these systems, the respective MTases contain an extended N‐terminus (approximately 70–90 aa), within which a clear helix‐turn‐helix (HTH) motif can be discerned.…”

The Methyltransferase from the LlaDII Restriction-Modification System Influences the Level of Expression of Its Own Gene

“…C.EcoO109I and its binding site exhibit no sequence similarity to the C-proteins and the C box, respectively. Another interesting mode of regulation was found for MspI (30), SsoII (10), and EcoRII (29). The MTases of these R-M systems contain N-terminal HTH motifs and have been found to interact directly with their own promoter sequences and to down-regulate the transcriptional activity.…”