Role and mechanism of action of C. PvuII, a regulatory protein conserved among restriction-modification systems.
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. 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. 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. 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. PvuII binding, overlapping the -10 promoter hexamer and the pvuIICR transcriptional starting points, is highly unusual for transcriptional activators.
Vijesurier
R M
RM
Center for Molecular Medicine, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Carlock
L
L
Blumenthal
R M
RM
Dunbar
J C
JC
D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism; D015257:DNA-Cytosine Methylases / Q000502:physiology*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000502:physiology*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D011514:Proteus vulgaris / Q000201:enzymology / Q000235:genetics; D013997:Time Factors; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; C047084:DNA modification methylase PvuII; D015257:DNA-Cytosine Methylases; C025630:CAGCTG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific