Non-Equilibrium Dynamics: Diffusion in Small Numbers and Ribosomal Self-Assembly

Abstract

Biological systems are encountered in states that are far from equilibrium. A change in the cell's condition triggers the flow of energy and matter that causes the cell's transition from that non-equilibrium state to a different state. Our interest is on non-equilibrium systems and the way these relate to the cell's limit as well as to the mechanisms of self-assembly. Cells contain proteins and nucleotides in numbers smaller than Avogadro's. In addition, advances in single-molecule experiments, which are, by definition, a case of the problem, have emphasized the importance of fluctuations. Does the result we get from a single-molecule measurement agree with what we would get from a bulk measurement? Is it a fluctuation from the mean? It is, thus, of biological interest to see the behavior of non-equilibrium systems at the limit where fluctuations become important. Using microfluidics, we concentrate on the diffusion of a small number of submicron particles in a system that is away from equilibrium. Therefore, we study the limit of Fick's Law, with special reference to the fluctuations that attend diffusive dynamics in order to experimentally test the theoretical predictions obtained via the use of E. T. Jaynes' principle of maximum caliber. The process of macromolecular self-assembly is also highly dynamical. The system's components come together, defeating in this way entropic effects, to form the system. In the case of the ribosome, whose importance lies in its ability to synthesize proteins, understanding the mechanism of the highly dimensional process of self-assembly becomes relevant when designing, for example, new antibiotics. The second part of this thesis concentrates on the RNA-protein interactions which, in the case of the ribosome, determine the mechanism of self-assembly. With the use of microfluidic technology and a fluorescence assay we determine the thermodynamics and kinetics of RNA folding and RNA-protein binding for a fragment of the bacterial 30S ribosomal subunit, paving the way for the study of the complete assembly of the 30S subunit.