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Journal of Bacteriology, January 2005, p. 488-497, Vol. 187, No. 2
0021-9193/05/$08.00+0     doi:10.1128/JB.187.2.488-497.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Nature of the Promoter Activated by C.PvuII, an Unusual Regulatory Protein Conserved among Restriction-Modification Systems

Department of Microbiology and Immunology and Program in Bioinformatics and Proteomics/Genomics, Medical College of Ohio, Toledo, Ohio

Received 8 June 2004/ Accepted 8 October 2004

A widely distributed family of small regulators, called C proteins, controls a subset of restriction-modification systems. The C proteins studied to date activate transcription of their own genes and that of downstream endonuclease genes; this arrangement appears to delay endonuclease expression relative to that of the protective methyltransferase when the genes enter a new cell. C proteins bind to conserved sequences called C boxes. In the PvuII system, the C boxes have been reported to extend from –23 to +3 relative to the transcription start for the gene for the C protein, an unexpected starting position relative to a bound activator. This study suggests that transcript initiation within the C boxes represents initial, C-independent transcription of pvuIICR. The major C protein-dependent transcript appears to be a leaderless mRNA starting farther downstream, at the initiation codon for the pvuIIC gene. This conclusion is based on nuclease S1 transcript mapping and the effects of a series of nested deletions in the promoter region. Furthermore, replacing the region upstream of the pvuIIC initiation codon with a library of random oligonucleotides, followed by selection for C-dependent transcription, yielded clones having sequences that resemble –10 promoter hexamers. The –35 hexamer of this promoter would lie within the C boxes. However, the spacing between C boxes/–35 and the apparent –10 hexamer can be varied by ±4 bp with little effect. This suggests that, like some other activator-dependent promoters, PpvuIICR may not require a –35 hexamer. Features of this transcription activation system suggest explanations for its broad host range.

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