Models of primitive cellular life: polymerases and templates in liposomes

Pierre-Alain Monnard () 0 1 Andrej Luptak 0 David W. Deamer 1 0 Department of Molecular Biology, Massachusetts General Hospital , Boston, MA 02114 , USA 1 Department of Chemistry and Biochemistry, University of California , Santa Cruz, CA 94720 , USA Nutrient transport, polymerization and expression of genetic information in cellular compartments are hallmarks of all life today, and must have appeared at some point during the origin and early evolution of life. Because the first cellular life lacked membrane transport systems based on highly evolved proteins, they presumably depended on simpler processes of nutrient uptake. Using a system consisting of an RNA polymerase and DNA template entrapped in submicrometre-sized lipid vesicles (liposomes), we found that the liposome membrane could be made sufficiently permeable to allow access of ionized substrate molecules as large as nucleoside triphosphates (NTPs) to the enzyme. The encapsulated polymerase transcribed the template-specific base sequences of the DNA to the RNA that was synthesized. These experiments demonstrate that units of genetic information can be associated with a functional catalyst in a single compartment, and that transcription of gene-sized DNA fragments can be achieved by relying solely on passive diffusion to supply NTPs substrates. 1. INTRODUCTION A fundamental property of life is polymer synthesis in a confined space, using free energy and nutrients available in the environment (Deamer et al. 1994; Walde et al. 1994; Deamer 1997; Tawfik & Griffiths 1998; Szostak et al. 2001). In contemporary cellular life, two polymersnucleic acids and proteinsare central to this process. In order to have the capacity for evolution, the two polymers must necessarily be linked through an information transfer process that allows genetic changes to be translated into altered phenotypes and then transmitted to ensuing generations. The origin of cellular life presumably occurred by self-assembly of organic compounds on the prebiotic Earth into encapsulated molecular systems capable of catalysed polymer synthesis. Although it is unknown whether nucleic acids and proteins were components of the first living systems, analogous polymers must have been synthesized by an yet unknown pathway, which were capable of the linked interactions that permit genetic variation and evolutionary selection of expressed phenotypes. Living cells are defined by lipid bilayer membranes, and liposomes have been explored previously as model protocells (Deamer & Oro 1980; Lazcano 1994a,b; Luisi et al. 1999; Szostak et al. 2001; Hanczyc & Szostak 2004). Investigations of enzymatic reactions within the boundaries of vesicles or liposomes have been performed in a variety of molecular systems (Chakrabarti et al. 1994; Walde et al. 1994; Oberholzer et al. 1995, 1999; Nomura et al. 2003; Ishikawa et al. 2004; Noireaux & Libchaber 2004; Chen et al. 2005). Two approaches to the design of these molecular systems have generally been followed. The first approach uses vesicular systems with diameters up to tens of micrometres to study coupled transcription and translation ( Nomura et al. 2003; Ishikawa et al. 2004; Noireaux & Libchaber 2004) or to encapsulate DNA / histone complexes ( Nomura et al. 2001). In the second approach, smaller vesicles in the submicrometre range were investigated to study reactions such as random RNA polymerization ( Walde et al. 1994), DNA amplification ( PCR; Oberholzer et al. 1995) and ribozyme cleavage (Chen et al. 2005). Although the size of primitive protocells is unknown, it seems probable that simple genetic/catalytic machinery could have fitted into a volume smaller than that of contemporary bacteria. A large membrane-defined volume, being relatively fragile, might have been disadvantageous in a natural environment, which is prone to sudden changes in pH, temperature or ionic strength. Furthermore, self-assembly of protocells was likely to have been a simple process. For example, in one plausible scenario, it is proposed that dried films of mixed lipid and polymers were rehydrated, producing large vesicles that would then be fragmented into smaller compartments (Hanczyc & Szostak 2004). Typical lipid bilayer systems have a significant limitation with respect to primitive cellular life, which is their relative impermeability to polar or ionic solutes required as substrates. To circumvent this limitation, in previous investigations, ionic substrates were typically encapsulated simultaneously with the enzyme machinery, ensuring an adequate substrate supply at least during the early stages of an encapsulated polymerization reaction (Oberholzer et al. 1995; Nomura et al. 2003; Ishikawa et al. 2004). In contemporary cells, the permeability barrier is essential for maintaining ionic concentration gradients that drive many bioenergetic processes. For this reason, complex systems of transport proteins that facilitate the movement of ions, nutrients and metabolites across the barrier are incorporated in biological membranes (Aidley & Stanfield 1996). The first forms of cellular life presumably lacked such systems and instead relied on simpler mechanisms, such as passive diffusion across their boundaries to accumulate nutrients from the environment (Deamer 1997). In order to provide a laboratory model of a protocell, we have developed a liposome system consisting of dimyristoylphosphatidylcholine (DMPC) vesicles with an encapsulated RNA transcription system composed of an enzyme, T7 RNA polymerase, its DNA template and magnesium ions. These vesicles have an average diameter in the submicrometre range. Substrate molecules for the transcription, nucleoside triphosphates (NTPs), are present in the external medium and must cross the bilayer barrier by passive diffusion to become available to the enzymatic system. In other words, the substrate molecules must diffuse through transient defects produced by disturbances in the lipid packing order (Paula et al. 1996). The frequency of such defects significantly increases at the lipid phase transition temperature ( Kanehisa & Tsong 1978; Mouritsen et al. 1995), an additional experimental variable. We also know that the bilayers of DMPC vesicles display a selective permeability barrier that permits permeation of monomers while retaining nucleotide dimers and higher oligomers (Monnard & Deamer 2001). This system allowed us to address the following questions: (i) How readily can macromolecules such as a polymerase enzyme and its template be captured within the volume of a single lipid vesicle with an average diameter in the submicrometre range? (ii) Can ionized substrates, such as NTPs, be made available to the polymerase at a rate sufficient to permit RNA synthesis? (iii) Does the microenvironment of a lipid vesicle affect the fidelity with which an encapsulated T7 polymerase transcribes a nucleotide sequence from a DNA template to RNA? In other words, how small can a compartment be with (...truncated)