): The ribosome, as the site where genetic information is translated into polypeptides which subsequently fold into proteins, and as the target site for antibiotic action, is an organelle with eminent public health relevance. X-ray crystallography and cryo-electron microscopy (cryo-EM) are the main tools to probe ribosomal structure. RNA has emerged as the most important component of the ribosome, acting as a structural scaffold and promoting most of the functionally important interactions with mRNA and tRNAs, as well as the peptidyl moiety. Understanding the dynamics of RNA in these interactions will be key to the understanding of ribosomal function. As atomic structures of rRNA becomes available, the exploration of RNA dynamics can be approached by a combination of genetic manipulations, cryo-EM, flexible fitting of X-ray structures, and molecular modeling and molecular dynamics. Pioneering work by this lab in the study of ribosome structure and function using cryo-EM, some of which were made in pursuing the goals in the previous funding period of this grant, position us well to take on some of these challenges. This renewal proposal describes a four-year research plan in collaboration with Dr. Steven Harvey (Specific Aim #1: atomic modeling of the decoding center and its dynamics), Dr. Albert Dahlberg (Specific Aim #2: point mutations to study switching behavior, and stabilizing functions of proteins), and Dr. Willy Wriggers (tools for fitting and docking of atomic structures into cryo-EM density maps).
Specific Aim #3 is the large-scale (non-atomic) modeling of the intersubunit ratchet movement during translocation observed by this group.
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