Cell cycle regulation of the tobacco ribonucleotide reductase small subunit gene is mediated by E2F-like elements

Ribonucleotide reductase (RNR) is a key enzyme involved in the DNA synthesi s pathway. The RNR-encoded genes are cell cycle regulated and specifically expressed in S phase. The promoter of the RNR2 gene encoding for the small subunit was isolated from tobacco. Both in vivo and in vitro studies of the DNA-protein interactions in synchronized BY2 tobacco cells showed that two E2F-like motifs were involved in multiple specific complexes, some of whic h displayed cell cycle-regulated binding activities. Moreover, these two el ements could specifically interact with a purified tobacco E2F protein. Inv olvement of the E2F elements in regulating the RNR2 promoter was checked by functional analyses in synchronized transgenic BY2 cells transformed with various RNR2 promoter constructs fused to the luciferase reporter gene. The two E2F elements were involved in upregulation of the promoter at the G1/S transition and mutation of both elements prevented any significant inducti on of the RNR promoter. In addition, one of the E2F elements sharing homolo gy with the animal E2F/cell cycle-dependent element motif behaved like a re pressor when outside of the S phase. These data provide evidence that E2F e lements play a crucial role in cell cycle regulation of gene transcription in plants.