Abstract
C-proteins control restriction–modification (R–M) systems’ genes transcription to ensure sufficient levels of restriction endonuclease to allow protection from foreign DNA while avoiding its modification by excess methyltransferase. Here, we characterize transcription regulation in C-protein dependent R–M system Kpn2I. The Kpn2I restriction endonuclease gene is transcribed from a constitutive, weak promoter, which, atypically, is C-protein independent. Kpn2I C-protein (C.Kpn2I) binds upstream of the strong methyltransferase gene promoter and inhibits it, likely by preventing the interaction of the RNA polymerase sigma subunit with the -35 consensus element. Diminished transcription from the methyltransferase promoter increases transcription from overlapping divergent C-protein gene promoters. All known C-proteins affect transcription initiation from R–M genes promoters. Uniquely, the C.Kpn2I binding site is located within the coding region of its gene. C.Kpn2I acts as a roadblock stalling elongating RNA polymerase and decreasing production of full-length C.Kpn2I mRNA. Mathematical modeling shows that this unusual mode of regulation leads to the same dynamics of accumulation of R–M gene transcripts as observed in systems where C-proteins act at transcription initiation stage only. Bioinformatics analyses suggest that transcription regulation through binding of C.Kpn2I-like proteins within the coding regions of their genes may be widespread.
Highlights
Type II restriction–modification (R–M) systems encode a restriction endonuclease that recognizes and cleaves specific DNA sequences and a methyltransferase that recognizes the same DNA sequence and methylates it first on one DNA strand - to produce hemimethylated DNA - and on the other strand to produce fully methylated DNA [1]
The genomes of many phages are devoid of recognition sites of R–M gene products commonly found in host bacteria [2], indicating that R–M systems have a profound influence on bacteriophages parasitizing on host bacteria
The pKpn2I plasmid contains the entire Kpn2I R–M system cloned on a low-copy number (15–20 copies per cell) pACYC vector [24]. pKpn2I is stably maintained in E. coli cells and provides resistance to phage infections [24], indicating that sufficient amounts of restriction endonuclease are produced
Summary
Type II restriction–modification (R–M) systems encode a restriction endonuclease that recognizes and cleaves specific DNA sequences and a methyltransferase that recognizes the same DNA sequence and methylates it first on one DNA strand - to produce hemimethylated DNA - and on the other strand to produce fully methylated DNA [1]. The genomes of many phages are devoid of recognition sites of R–M gene products commonly found in host bacteria [2], indicating that R–M systems have a profound influence on bacteriophages parasitizing on host bacteria. While this beneficial property doubtless contributed to wide dissemination of R–M systems in the eubacterial kingdom [3,4], multiple functions unrelated to phage defense may have played a role (see [5] for review). Premature appearance of endonuclease activity upon entry of a genetic element carrying R–M system genes into a naıve host will lead to host DNA degradation [7,8].
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