In this study, RLM-RACE was used to identify the transcriptional start site 387 bp upstream of the translational start. Evolutionarily conserved transcription factor binding sites were identified, and a series of luci...In this study, RLM-RACE was used to identify the transcriptional start site 387 bp upstream of the translational start. Evolutionarily conserved transcription factor binding sites were identified, and a series of luciferase reporter constructs driven by BMPR2 promoter elements used to determine their functional relevance. We found the promoter area from 983 bp to 90 bp upstream of the transcriptional start gave maximal activity, greater than longer constructs, with an area between 570 bp and 290 bp upstream of the transcriptional start containing an important repressor element. To characterize this repressor, we used a combination of EMSA, mutation of the EGR1 binding site, transfection with EGR1 and NAB1 constructs, and mutation of the NAB1 binding site within the EGR1 protein. From this we conclude that EGR1 is essential to BMPR2 transcription, but that NAB1 binding to EGR1 causes it to act as a repressor.展开更多
The unicellular green alga Chlamydomonas reinhardtii is capable of using organic and inorganic carbon sources simultaneously, which requires the adjustment of photosynthetic activity to the prevailing mode of carbon a...The unicellular green alga Chlamydomonas reinhardtii is capable of using organic and inorganic carbon sources simultaneously, which requires the adjustment of photosynthetic activity to the prevailing mode of carbon assimilation. We obtained novel insights into the regulation of light-harvesting at photosystem II （PSII） following altered carbon source avail- ability. In C. reinhardtii, synthesis of PSll-associated light-harvesting proteins （LHCBMs） is controlled by the cytosolic RNA- binding protein NAB1, which represses translation of particular LHCBM isoform transcripts. This mechanism is fine-tuned via regulation of the nuclear NAB1 promoter, which is activated when linear photosynthetic electron flow is restricted by CO2- limitation in a photoheterotrophic context. In the wild-type, accumulation of NAB1 reduces the functional PSII antenna size, thus preventing a harmful overexcited state of PSII, as observed in a NABl-less mutant. We further demonstrate that trans- lation control as a newly identified long-term response to prolonged CO2-1imitation replaces LHCII state transitions as a fast response to PSII over-excitation. Intriguingly, activation of the long-term response is perturbed in state transition mutant stt7, suggesting a regulatory link between the long- and short-term response. We depict a regulatory circuit operating on distinct timescales and in different cellular compartments to fine-tune light-harvesting in photoheterotrophic eukaryotes.展开更多
文摘In this study, RLM-RACE was used to identify the transcriptional start site 387 bp upstream of the translational start. Evolutionarily conserved transcription factor binding sites were identified, and a series of luciferase reporter constructs driven by BMPR2 promoter elements used to determine their functional relevance. We found the promoter area from 983 bp to 90 bp upstream of the transcriptional start gave maximal activity, greater than longer constructs, with an area between 570 bp and 290 bp upstream of the transcriptional start containing an important repressor element. To characterize this repressor, we used a combination of EMSA, mutation of the EGR1 binding site, transfection with EGR1 and NAB1 constructs, and mutation of the NAB1 binding site within the EGR1 protein. From this we conclude that EGR1 is essential to BMPR2 transcription, but that NAB1 binding to EGR1 causes it to act as a repressor.
文摘The unicellular green alga Chlamydomonas reinhardtii is capable of using organic and inorganic carbon sources simultaneously, which requires the adjustment of photosynthetic activity to the prevailing mode of carbon assimilation. We obtained novel insights into the regulation of light-harvesting at photosystem II （PSII） following altered carbon source avail- ability. In C. reinhardtii, synthesis of PSll-associated light-harvesting proteins （LHCBMs） is controlled by the cytosolic RNA- binding protein NAB1, which represses translation of particular LHCBM isoform transcripts. This mechanism is fine-tuned via regulation of the nuclear NAB1 promoter, which is activated when linear photosynthetic electron flow is restricted by CO2- limitation in a photoheterotrophic context. In the wild-type, accumulation of NAB1 reduces the functional PSII antenna size, thus preventing a harmful overexcited state of PSII, as observed in a NABl-less mutant. We further demonstrate that trans- lation control as a newly identified long-term response to prolonged CO2-1imitation replaces LHCII state transitions as a fast response to PSII over-excitation. Intriguingly, activation of the long-term response is perturbed in state transition mutant stt7, suggesting a regulatory link between the long- and short-term response. We depict a regulatory circuit operating on distinct timescales and in different cellular compartments to fine-tune light-harvesting in photoheterotrophic eukaryotes.
