Talk abstracts

Talk on Saturday 11:00-11:15am submitted by James Marks

tRNA dynamics during antibiotic dependent translation arrest

James Marks (University of Illinois at Chicago), Junhong Choi (Stanford University), Nora Vazquez-Laslop (University of Illinois at Chicago), Joseph Puglisi (Stanford University), Alexander Mankin (University of Illinois at Chicago)

Chloramphenicol and linezolid are inhibitors of bacterial protein synthesis. They bind to the A-site of the peptidyl transferase center in the ribosome, the site occupied by all incoming aminoacyl-tRNAs during translation elongation. Therefore it was thought that chloramphenicol and linezolid inhibit formation of every peptide bond because they interfere with the accommodation of any aminoacyl-tRNA. However, we have recently shown that chloramphenicol and linezolid are not universal inhibitors but, instead, their action depends on the amino acid sequence of the nascent peptide. Specifically, translation is strongly arrested when an alanine is in the penultimate position of the nascent chain. To investigate how antibiotic dependent arrest influences translation dynamics we used single-molecule FRET to discretely monitor ribosome and tRNA conformations in real-time during translation of model templates in the presence or absence of chloramphenicol. We found that, in spite of the presence of chloramphenicol, the ribosome progressed normally when translating the non-arrest template. In contrast, a ribosome translating an arrest peptide in the presence of chloramphenicol binds A-site tRNA but in a non-productive fashion because it remains in a partially accommodated state for an extended period of time. After the non-productive A-site tRNA finally dissociates from the ribosome, another round of non-productive binding can occur. Our findings not only contribute to understanding the mechanism of action of ribosomal antibiotics but also reveal critical aspects tRNA dynamics in the peptidyl transferase center during protein synthesis.

Keywords: Ribosome, Antibiotic, tRNA