Protein synthesis and active transport of vesicular cargoes are vital to development of all tissues and to the targeted delivery of organelles, proteins, and signaling molecules in eukaryotes. Accordingly, defects in protein expression and transport are linked to developmental, neurodegenerative, pigmentation, immunological, and other diseases. Knowing the detailed mechano-chemistry and structural dynamics of the ribosome and motor proteins is essential for understanding and interpreting their roles in the cell. We developed a number of powerful new biophysical tools that reveal the modulation of structural dynamics and reaction kinetics of the protein synthesis elongation cycle and molecular motors under applied mechanical force, discriminate models of energy transduction and elucidate the essential rotational motions of conformational transitions of specific domains within the elongation factors and motor proteins. We will apply these unique tools to investigate the rhythm of protein synthesis in bacteria and eukaryotes and mechanisms that functionally modulate it and the divergent biochemical and mechanical properties of myosins-I, V, VI and X, and dynein isoforms. Understanding functional properties that have that have not yet been approached at the mechanistic detail is now possible. This application combines and extends three NIGMS grants, and so has a number of aims.
Our Aims i n protein synthesis are to Determine the mechanisms of modulation of protein synthesis rate of 1) downstream mRNA 2o structure and 2) upstream Shine-Delgarno (SD) sequences.
Aim 3) is to Develop a eukaryotic single molecule FRET platform for detailed study of peptide elongation in higher organisms.
Aim 4) is to Define how read-through of premature stop codons takes place and how small molecule enhancers of read-through operate, which are promising therapies in many diseases, e.g. Duchenne muscular dystrophy and cyctic fibrosis. For molecular motors, for each of the target myosins I, V, VI and X, to 5) test the thermal search hypothesis, determine their flexibility and solve the mechanisms of their high directionality; 6) Determine the mechanical force-dependence of association of motors with their cytoskeletal tracks and the energetics of their mechanical stroke using an ultra-high-speed optical trap; 7) Determine the dynamics of ATP association and ADP and Pi dissociation from the motors under force using combined optical trapping and single molecule TIRF microscopy; 8) Determine the rotational motions of motor heads and lever arms that generate force and cargo translocation using single molecule polarized TIRF microscopy; 9) Determine motor force and force regulation on intracellular cargos, and 10) Determine how many kinesin motors are actively engaged on intracellular cargos and their spatial distribution around the cargo surface. Overall, these studies will lead to a much more general view of the mechanisms and characteristics of the ribosome and molecular motors in vitro and in live cells leading to a more rigorous understanding of their functions in cell biology and disease.
Protein synthesis on the ribosome and intracellular motility and cell division, based on the molecular motors are crucial for development and maintenance of all of the organs in the body. The specific cellular machines to be studied here, the ribosome, myosins, kinesin and cytoplasmic dyneins, are required for appropriate neuronal development, sensorineural function in hearing and eyesight, the immune response and cell division. Thus errors of expression or function lead to severe developmental neurological, cardiovascular, immunological deficits, and play roles in cancer cell division and metastasis, muscular dystrophy, cystic fibrosis and many other diseases. The studies proposed here will give fundamental information on how gene expression, cellular development, and transport motors function and may eventually lead to therapeutic targets in these diseases.
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