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Journal of Bacteriology, October 2007, p. 6928-6935, Vol. 189, No. 19
0021-9193/07/$08.00+0     doi:10.1128/JB.00127-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Negative Regulation of the EcoRI Restriction Enzyme Gene Is Associated with Intragenic Reverse Promoters ^,

Yaoping Liu^1,2 and Ichizo Kobayashi^1,2,3*

Department of Medical Genome Sciences, Graduate School of Frontier Science, University of Tokyo, Tokyo, Japan,¹ Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan,² Graduate Program in Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Tokyo, Japan³

Received 25 January 2007/ Accepted 26 June 2007

Type II restriction-modification systems are expected to possess mechanisms for tight regulation of their expression to suppress the potential of lethal attack on their host bacteria when they establish and maintain themselves within them. Although the EcoRI restriction enzyme has been well characterized, regulation of its expression is still poorly understood. In this study, mutational analysis with lacZ gene fusion and primer extension assay identified a promoter for the transcription of the ecoRIR gene. Further analyses revealed that an intragenic region containing two overlapping reverse promoter-like elements acted as a negative regulator for ecoRIR gene expression. The activity of these putative reverse promoters was verified by transcriptional gene fusion, primer extension and in vitro transcription. Mutations in these reverse promoters resulted in increased gene expression in both translational and transcriptional gene fusions. An RNase protection assay revealed that the transcript level of the wild type relative to that of the reverse promoter mutant at the downstream regions was much lower than the level at the upstream regions. This suggests that these reverse promoter-like elements affect their downstream transcript level. The possible mechanisms of this kind of negative regulation, in addition to their possible biological roles, are discussed.

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* Corresponding author. Mailing address: Department of Medical Genome Sciences, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Fax: 81 3 5449 5422. E-mail: ikobaya{at}ims.u-tokyo.ac.jp

Published ahead of print on 6 July 2007.

Supplemental material for this article may be found at http://jb.asm.org/.

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Journal of Bacteriology, October 2007, p. 6928-6935, Vol. 189, No. 19
0021-9193/07/$08.00+0     doi:10.1128/JB.00127-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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