? We propose to use biochemical and biophysical approaches to understand the mechanism of action of the ribosome in terms of its molecular structure. Now that the three-dimensional structure of the ribosome is known, we wish to understand the many molecular interactions and movements that occur during translation, particularly those that involve ribosomal RNA. We will focus on the specific problems of (1) How the ribosome discriminates initiator tRNA from elongator tRNA during translational initiation; (2) The roles of the extended protein """"""""tails"""""""" in the P site of the 30S subunit; (3) The molecular basis of coupled translocation of mRNA and tRNA during translation and of the accommodation of aminoacyl-tRNA; and (4) How the ribosome unwinds secondary structure in mRNA during translation. To address these questions, we will use a range of biochemical approaches, including chemical probing, toeprinting and in vitro reconstitution from a complete set of recombinant 30S proteins. To study ribosomal dynamics and their associated atomic forces, we will implement several biophysical approaches (in collaboration with three different biophysical groups), including the use of real-time hydroxyl radical probing of rRNA using synchrotron radiation, fluorescence resonance energy transfer, and optical tweezers. ? ?
Showing the most recent 10 out of 69 publications
