The asymmetric localization of mRNA plays an important role in coordinating posttranscriptional events in eukaryotic cells. We investigated the peripheral mitochondrial localization of nuclear-encoded mRNAs (MLR) in various conditions in which the mRNA binding protein context and the translation efficiency were altered. We identified Puf3p, a Pumilio family RNA-binding protein, as the first trans-acting factor controlling the MLR phenomenon. This allowed the characterization of two classes of genes whose mRNAs are translated to the vicinity of mitochondria. Class I mRNAs (256 genes) have a Puf3p binding motif in their 3'UTR region and many of them have their MLR properties deeply affected by PUF3 deletion. Conversely, mutations in the Puf3p binding motif alter the mitochondrial localization of BCS1 mRNA. Class II mRNAs (224 genes) have no Puf3p binding site and their asymmetric localization is not affected by the absence of PUF3. In agreement with a co-translational import process, we observed that the presence of puromycin loosens the interactions between most of the MLR-mRNAs and mitochondria. Unexpectedly, cycloheximide, supposed to solidify translational complexes, turned out to destabilize a class of mRNA-mitochondria interactions. Classes I and II mRNAs, which are therefore transported to the mitochondria through different pathways, correlated with different functional modules. Indeed, Class I genes code principally for the assembly factors of respiratory chain complexes and the mitochondrial translation machinery (ribosomes and translation regulators). Class II genes encode proteins of the respiratory chain or proteins involved in metabolic pathways. Thus, MLR, which is intimately linked to translation control, and the activity of mRNA-binding proteins like Puf3p, may provide the conditions for a fine spatiotemporal control of mitochondrial protein import and mitochondrial protein complex assembly. This work therefore provides new openings for the global study of mitochondria biogenesis.
Gene expression plays a central role in the orchestration of cellular processes. The use of inducible promoters to change the expression level of a gene from its physiological level has significantly contributed to the understanding of the functioning of regulatory networks. However, from a quantitative point of view, their use is limited to short-term, population-scale studies to average out cell-to-cell variability and gene expression noise and limit the nonpredictable effects of internal feedback loops that may antagonize the inducer action. Here, we show that, by implementing an external feedback loop, one can tightly control the expression of a gene over many cell generations with quantitative accuracy. To reach this goal, we developed a platform for real-time, closed-loop control of gene expression in yeast that integrates microscopy for monitoring gene expression at the cell level, microfluidics to manipulate the cells' environment, and original software for automated imaging, quantification, and model predictive control. By using an endogenous osmostress responsive promoter and playing with the osmolarity of the cells environment, we show that long-term control can, indeed, be achieved for both time-constant and time-varying target profiles at the population and even the single-cell levels. Importantly, we provide evidence that real-time control can dynamically limit the effects of gene expression stochasticity. We anticipate that our method will be useful to quantitatively probe the dynamic properties of cellular processes and drive complex, synthetically engineered networks.model based control | computational biology | high osmolarity glycerol pathway | quantitative systems biology U nderstanding the information processing abilities of biological systems is a central problem for systems and synthetic biology (1-6). The properties of a living system are often inferred from the observation of its response to static perturbations. Timevarying perturbations have the potential to be much more informative regarding the dynamics of cellular functions (7-12). Currently, it is not possible to precisely perturb protein levels in an analogous manner, even though this perturbation would be instrumental in our understanding of gene regulatory networks. Indeed, despite the development of novel regulatory systems, including various RNA-based solutions (13), transcriptional control by means of inducible promoters is still the preferred method for manipulating protein levels (14, 15). Unfortunately, inducible promoters have several generic limitations. First, there is a significant delay between gene expression activation and effective protein synthesis. Second, many cellular processes can interfere with gene expression through internal feedback loops whose effects are hard to predict. Third, the process of gene expression shows significant levels of noise (16)(17)(18). Given these limitations, novel experimental strategies are required to gain quantitative, real-time control of gene expression in vivo.Here, we see the...
The Saccharomyces cerevisiae PDR3 gene, located near the centromere of chromosome II, has been completely sequenced and characterised. Mutations pdr3-1 and pdr3-2, which confer resistance to several antibiotics can be complemented by a wild-type allele of the PDR3 gene. The sequence of the wild-type PDR3 gene revealed the presence of a long open reading frame capable of encoding a 976-amino acid protein. The protein contains a single Zn(II)2Cys6 binuclear-type zinc finger homologous to the DNA-binding motifs of other transcriptional activators from lower eukaryotes. Evidence that the PDR3 protein is a transcriptional activator was provided by demonstrating that DNA-bound LexA-PDR3 fusion proteins stimulate expression of a nearby promoter containing LexA binding sites. The use of LexA-PDR3 fusions revealed that the protein contains two activation domains, one localised near the N-terminal, cysteine-rich domain and the other localised at the C-terminus. The salient feature of the PDR3 protein is its similarity to the protein coded by PDR1, a gene responsible for pleiotropic drug resistance. The two proteins show 36% amino acid identity over their entire length and their zinc finger DNA-binding domains are highly conserved. The fact that the absence of both PDR1 and PDR3 (simultaneous disruption of the two genes) enhances multidrug sensitivity strongly suggests that the two transcriptional factors have closely related functions.
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