| We have achieved two goals in this thesis: (1) Development of single molecule (SM) optical apparatii; (2) Systematic studies of the folding of large RNA with SM methods. SM FRET (Fluorescence resonance energy transfer) and comparative ensemble measurements are performed to study the equilibrium folding of the catalytic domain of Bacterial RNase P, a large (255nt) ribozyme. A two-photon microscope with 400nm lateral resolution based on a 800nm 40fs pulsed Ti:Sapphire laser was built for bioimaging and SM lifetime measurements. Another confocal apparatus was built for SM dual-color, FRET and polarization and fluorescence correlation spectroscopy (FCS). Computerized SM optical detection schemes are developed.; Three protocols were implemented to doubly label RNase P RNA, and several FRET pairs were investigated. We explore a number of factors, including Mg 2+ concentration, metal ions, FRET pair locations, P Protein cofactor to study two homolog RNA, mesophilic and thermophilic C domain. Based on SM trajectories and FRET histograms, we conclude that the folding of the mesophilic C domain is multi-state and evaluate its folding free energy landscape. A new level of heterogeneity is observed for thermophilic C domain. It can be approximated as a two-state system when the [Mg2+] is far below the equilibrium midpoint, KMg. Close examination of SM FRET trajectories reveals four (FRET) states at and above K Mg with a high degree of connectivity. We observe two dynamics behaviors for the four-state mechanism: a slow phase characterized by dwell times in the seconds to minutes range and a fast dynamics phase with millisecond fluctuations. We also observe a divalent cation (Mg2+, Ca 2+, Sr2+, Ba2+) dependence of the two parallel folding pathways to the native structure. A detailed folding mechanism for the thermophilic C domain is proposed. The dwell time distributions for the thermophilic C domain span at least three decades and obey a non-exponential (close to power law) behavior, supporting the notion that there is a distribution of barriers and that no barrier/rate dominates the interconversion between any two states. As a proposal for future study, we have discovered conformational fluctuations of the thermophilic C domain RNase P to be very different in the presence of the P Protein cofactor. To understand the interactions between functional RNA and its various cofactors will shed light on the origin of the evolution-optimized molecular machinery. |
