Cellular genes that are functionally related to each other are usually confined in specialized subcellular compartments for efficient biochemical reactions. Construction of spatially controlled biosynthetic systems will facilitate the study of biological design principles. Herein, we fabricated a gene circuit compartment by coanchoring two functionrelated genes on surface of gold nanoparticles and investigated the compartment effect on cascade gene expression in a cell-free system. The gene circuit consisted of a T7 RNA polymerase (T7 RNAP) expression cassette as regulatory gene and a fluorescent protein expression cassette as regulated reporter gene. Both the expression cassettes were attached on a Y-shaped DNA nanostructure whose other two branches were mercapto-modified in order to steadily anchor the gene expression cassettes on the surface of gold nanoparticles. Experimental results demonstrated that both the yield and initial expression rate of the fluorescent reporter protein in the gene circuit compartment system were enhanced compared with those in free gene circuit system. Mechanism investigation revealed that the gene circuit compartment on nanoparticle made the regulatory gene and regulated reporter gene spatially proximal at nanoscale, thus effectively improving the transfer efficiency of the regulatory proteins (T7 RNAP) from regulatory genes to the regulated reporter genes in the compartments, and consequently, the biochemical reaction efficiency was significantly increased. This work not only provided a simplified model for rational molecular programming of genes circuit compartments on nanointerface but also presented implications for the cellular structure−function relationship.
Protein kinases are dynamic molecular switches that sample multiple conformational states. The regulatory subunit of PKA harbors two cAMP-binding domains [cyclic nucleotide-binding (CNB) domains] that oscillate between inactive and active conformations dependent on cAMP binding. The cooperative binding of cAMP to the CNB domains activates an allosteric interaction network that enables PKA to progress from the inactive to active conformation, unleashing the activity of the catalytic subunit. Despite its importance in the regulation of many biological processes, the molecular mechanism responsible for the observed cooperativity during the activation of PKA remains unclear. Here, we use optical tweezers to probe the folding cooperativity and energetics of domain communication between the cAMP-binding domains in the apo state and bound to the catalytic subunit. Our study provides direct evidence of a switch in the folding-energy landscape of the two CNB domains from energetically independent in the apo state to highly cooperative and energetically coupled in the presence of the catalytic subunit. Moreover, we show that destabilizing mutational effects in one CNB domain efficiently propagate to the other and decrease the folding cooperativity between them. Taken together, our results provide a thermodynamic foundation for the conformational plasticity that enables protein kinases to adapt and respond to signaling molecules.
The physical distance between genes plays important roles in controlling gene expression reactions in vivo. Herein, we report the design and synthesis of a branched gene architecture in which three transcription units are integrated into one framework through assembly based on the polymerase chain reaction (PCR), together with the exploitation of these constructs as “gene compartments” for cell‐free gene expression reactions, probing the impact of this physical environment on gene transcription and translation. We find that the branched gene system enhances gene expression yields, in particular at low concentrations of DNA and RNA polymerase (RNAP); furthermore, in a crowded microenvironment that mimics the intracellular microenvironment, gene expression from branched genes maintains a relatively high level. We propose that the branched gene assembly forms a membrane‐free gene compartment that resembles the nucleoid of prokaryotes and enables RNAP to shuttle more efficiently between neighboring transcription units, thus enhancing gene expression efficiency. Our branched DNA architecture provides a valuable platform for studying the influence of “cellular” physical environments on biochemical reactions in simplified cell‐free systems.
Cellular physical microenvironment such as crowding shows great influence on enzymatic reactions. Herein, we report a new finding that saccharides with low molecular weight create an effective crowding microenvironment for gene expression in cell-free protein synthesis, which provides valuable implications for living systems. Four saccharides including sorbose, galactose, sucrose, and cellobiose are screened out as effective crowders. At a low concentration range of saccharides, both the mRNA and protein amounts present an upward trend with the concentration increment of saccharides; when the concentrations exceed a critical value, the mRNA and protein amounts decrease. A mechanism is proposed that at low concentrations of saccharides, the effective concentrations of reactants increase due to the coexistence of crowders and reactants in a finite volume; when the concentrations exceed a critical value, the molecular diffusion of reactants is dominantly restricted due to the increased viscosity. Our finding opens a new view that saccharides with low molecular weight could be crowders and provides a new insight that substances with low molecular weight in cells would produce a crowding effect on biochemical reactions in living systems.
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