RESUMO
E2F factors are implicated in various cellular processes including specific gene induction at the G1/S transition of the cell cycle. We present in this study a novel regulatory aspect for the tobacco large subunit of ribonucleotide reductase (R1a) and its encoding gene (RNR1a) in the UV-C response. By structural analyses, two E2F sites were identified on the promoter of this gene. Functional analysis showed that, in addition to their role in the specific G1/S induction of the RNR1a gene, both E2F sites were important for regulating specific RNR1a gene expression in response to UV-C irradiation in non-synchronized and synchronized cells. Concomitantly, western blot and cellular analyses showed an increase of a 60 kDa E2F factor and a transient translocation of a GFP-R1a protein fusion from cytoplasm to nucleus in response to UV irradiation.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica de Plantas , Nicotiana/enzimologia , Nicotiana/genética , Ribonucleotídeo Redutases/genética , Fatores de Transcrição/fisiologia , Raios Ultravioleta , Transporte Ativo do Núcleo Celular , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Núcleo Celular/enzimologia , Replicação do DNA , Fatores de Transcrição E2F , Regulação Enzimológica da Expressão Gênica , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Subunidades Proteicas/análise , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Elementos de Resposta , Ribonucleotídeo Redutases/análise , Ribonucleotídeo Redutases/metabolismo , Fase S , Nicotiana/citologia , Nicotiana/efeitos da radiação , Ativação TranscricionalRESUMO
The RB/E2F pathway is involved in the control of the G(1)/S transition of the eukaryotic cell cycle where various S phase genes are activated by specific E2F factors. Ribonucleotide reductase (RNR) plays an essential role in the DNA synthesis pathway. Earlier studies showed that there are at least two RNR1 genes (RNR1a and RNR1b) and one RNR2 gene in tobacco. In synchronized tobacco BY2 cells, RNR1b gene expression is at its highest level in S phase. To investigate transcriptional regulation of the RNR1b gene, its promoter region was cloned and sequenced. Unlike its animal counterparts, the tobacco RNR1b promoter contains a consensus E2F-binding site. Surprisingly, this site is found in the leader sequence of the gene. We show here by gel shift analysis and antibody competition that one nuclear complex specifically binds this motif, and an E2F factor is part of this complex. Using reporter gene analysis, tobacco RNR1b promoter activity was detected during S phase in synchronized cells and in plant meristematic tissues. Mutation of the E2F element substantially reduced both activities. For the first time in plants, a single E2F motif found in the leader sequence plays an important role in the meristem and S phase-specific expression of the tobacco RNR1b gene.
