Molecular mechanisms of translation termination in bacteria

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Translation termination - an essential biological process in all unicellular and multicellular organisms - ensures that all proteins manufactured by the molecular machine known as the ribosome have the lengths strictly defined by the genes that code for them. This process is mediated by proteins know as Release Factors, which promote release of newly synthesized polypeptide product from the ribosome. Release must be tightly controlled to prevent the accumulation of truncated or extended polypeptides that can be non-functional or even toxic to the cell. The goals of the current project are to elucidate the molecular mechanism implemented by the Release Factors during the fundamental biological process of termination of protein synthesis. This project will have a broad impact on the advancement of our understanding of biology at the molecular level. Ultimately, the mechanistic details of the ribosomal machinery to be determined can reveal new avenues for the development of antibiotics that target the bacterial ribosome. In addition, the project will train both undergraduate and graduate students in powerful techniques in structural biology and introduce high school students to research at the forefront of structural and molecular biology. All Release Factors (RF) share a universally conserved tripeptide sequence motif Gly-Gly-Gln (GGQ) that is critical for eliciting hydrolytic activity at the active site of the ribosome, the peptidyl transferase center. Despite a number of previous biochemical and structural studies, the exact molecular mechanism of how Release Factors promote hydrolysis of peptidyl-tRNA at the peptidyl transfer center remains elusive. Thus, the overall goal of the proposed project is to uncover this yet undefined catalytic mechanism of RF-mediated peptide release and answer the long-standing fundamental questions in the field: how do release factors promote hydrolysis of the peptidyl-tRNA in the ribosome and what is the contribution of the universally conserved GGQ tripeptide motif to this process? As part of this work, multiple crystal structures of pre-termination 70S-RF complexes will be determined. These structures require the preparation of 70S-RF complexes trapped in the pre-termination state with the use non-hydrolyzable amide-linked peptidyl-tRNAs that will be engineered.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.