Role and Mechanism of Action of C · Pvu II, a Regulatory Protein Conserved among Restriction-Modification Systems

Abstract

The PvuII restriction-modification system is a type II system, which means that its restriction endonuclease and modification methyltransferase are independently active proteins. The PvuII system is carried on a plasmid, and its movement into a new host cell is expected to be followed initially by expression of the methyltransferase gene alone so that the new host's DNA is protected before endonuclease activity appears. Previous studies have identified a regulatory gene (pvuIIC) between the divergently oriented genes for the restriction endonuclease (pvuIIR) and modification methyltransferase (pvuIIM), with pvuIIC in the same orientation as and partially overlapping pvuIIR. The product of pvuIIC, C ⅐ PvuII, was found to act in trans and to be required for expression of pvuIIR. In this study we demonstrate that premature expression of pvuIIC prevents establishment of the PvuII genes, consistent with the model that requiring C ⅐ PvuII for pvuIIR expression provides a timing delay essential for protection of the new host's DNA. We find that the opposing pvuIIC and pvuIIM transcripts overlap by over 60 nucleotides at their 5 ends, raising the possibility that their hybridization might play a regulatory role. We furthermore characterize the action of C ⅐ PvuII, demonstrating that it is a sequence-specific DNA-binding protein that binds to the pvuIIC promoter and stimulates transcription of both pvuIIC and pvuIIR into a polycistronic mRNA. The apparent location of C ⅐ PvuII binding, overlapping the ؊10 promoter hexamer and the pvuIICR transcriptional starting points, is highly unusual for transcriptional activators.The bacterial type II restriction-modification systems include a DNA modification methyltransferase (MTase) and a restriction endonuclease (REase), both of which act independently on the same DNA sequence (65). The REase cleaves duplex DNA sequences in the absence of sequence-specific DNA modification by the MTase. These systems can defend bacterial cells against viral infection, although other functional roles have also been proposed (45). Restriction-modification systems have provided an important focus for studies of molecular recognition. Biochemical and crystallographic analyses are yielding significant insights into the mechanisms of sequence recognition and catalytic activity of these proteins (3,18,50,66).

Mobility of a Restriction-Modification System Revealed by Its Genetic Contexts in Three Hosts

“…1 show that the bidirectional genetic organization of the PvuII and SptAI systems is conserved, with the methyltransferase gene diverging from the genes for the regulator and endonuclease (which are cotranscribed in the PvuII system) (73). Figure 2 compares the amino acid sequences of the corresponding open reading frames (ORFs) from these two systems.…”

“…We routinely search the microbial genome database (www.ncbi.nlm.nih.gov/cgi-bin/Entrez/genom_table_cgi) by using the NCBI BLAST server (2,29) in an attempt to find new members of the C protein family of transcriptional regulators among RM systems (3,11,27,59,68,69,72,73). One such match, in S. enterica serovar Paratyphi A (Genome Sequencing Center, personal communication; genome.wustl.edu /gsc/Projects/bacterial/paratyphi/paratyphi.shtml), was flanked by methyltransferase and restriction endonuclease genes, both of which surprisingly showed very close relationship to another RM system cloned in this laboratory-PvuII (10).…”

“…The regulation of this system has been studied and involves an autogenous regulatory protein with unusual properties, called C•PvuII (1,68,69,73). PvuII is also the first RM system for which X-ray crystallography yielded structures for both the endonuclease and the methyltransferase (5,16,22).…”

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

“…One group, exemplified by PvuII (31) and BamHI (8), is regulated at the transcriptional level by small so-called Cproteins; the genes that encode these proteins are usually located upstream of and in some cases partially overlap the REase gene. The C-proteins constitute a family of regulatory proteins that bind to a common operator sequence (C box) through a helix-turn-helix (HTH) motif (26,32,33). They function as transcriptional activators of the REase genes, as well as their own genes, and in some systems, such as BamHI, they have been found to down-regulate expression of the MTase genes as well and thus to have dual functions.…”

Maintenance Forced by a Restriction-Modification System Can Be Modulated by a Region in Its Modification Enzyme Not Essential for Methyltransferase Activity

“…One regulatory mechanism employs a small protein, the so-called C protein (49,66). The C proteins specifically bind a DNA operator sequence to regulate expression of the restriction enzyme, modification enzyme, or both (70). This group of proteins is likely to delay the expression of the restriction enzyme to prevent cell death during establishment in a new host (49).…”