The overall goals of this project are to study the dynamics of ribosome movement along mRNA, the penultimate step in protein assembly, and to progress towards a clearer understanding of the processes that control this movement. Knowledge of the details of protein assembly will further the development of genetically based pharmaceuticals and thereby improve human health. Towards this end we will use total internal reflection fluorescence (TIRF) video microscopy to study folding dynamics of mRNA in real time using nanometer-resolved localization of quantum dot fluorescent DMA probes placed strategically along the mRNA. To vary the dynamics microfluidic systems will be used for precise delivery of reagents and for accurate control of temperature. A novel trip-wire probe methodology will be used to study ribosome/mRNA interactions. Quantum dot probes conjugated to short mRNA sequences will be dislodged as the ribosome passes over them. The resulting loss of fluorescence establishes position and allows precise time measurement with 0.1 sec resolution. Multiple probes placed strategically along the mRNA will allow detailed examination of ribosome/RNA interactions. With the multiple probe technology we will study the effect of mRNA secondary and tertiary structure on ribosome processivity and translation kinetics. [unreadable] [unreadable]