Talk abstracts

Talk on Friday 04:15-04:30pm submitted by May Daher

Exploration of the Ribosome’s Rescue Mechanism By Single Molecule Fluorescence Resonance Energy Transfer

May Daher Farhat (Chemistry Department, Wayne State University), David Rueda (Chemistry Department, Wayne State University)

Abstract:
Truncated mRNAs missing a stop codon cause synthesis of defective proteins and trap ribosomes at the end of their incomplete message. The stalled ribosome causes 2 major problems: first, a non-functional protein, which can be toxic for the cell; second, depletion of the pool of ribosomes available for translation. In bacteria, these problems can be resolved by a rescue process called trans-translation, which employs a noncoding RNA and its cofactor SmpB. Previous studies showed the importance of tmRNA during gene expression and for virulence in many pathogenic bacteria, making it an attractive target for antimicrobial drug discovery. However, key aspects of this mechanism including how the tmRNA-SmpB complex enters into and interacts with stalled ribosomes are not fully understood.
We are developing a Single Molecule Fluorescence Resonance Energy Transfer (smFRET) assay to investigate how this complex identifies and binds the stalled ribosome. We have labeled P-site phe-tRNA with a FRET donor and the 5’ end of the tmRNA open reading frame with a FRET acceptor. Our preliminary data show multiple FRET states indicating that tmRNA binding and accommodation into the ribosome. We are also labeling SmpB to further characterize the tmRNA trans-translation mechanism.

Keywords: tmRNA, SmpB, Single molecule