Talk abstracts
Talk on Friday 03:45-04:00pm submitted by Chathuri Pathirage
Dihydrouridine modification in tRNA impact ribosome frame maintenance during protein synthesis
Chathuri Pathirage (Department of Chemistry, University of Michigan), Kaley Simcox (Department of Chemistry, University of Michigan), Maddy Zamecnik (Department of Chemistry, University of Michigan), Xingchen Liu (Department of Chemistry, University of Michigan), Lauren Barnes, Yubin Kim (Department of Chemistry, University of Michigan), Kristin S. Koutmou (Department of Chemistry, University of Michigan)
Abstract:
tRNAs are the most modified of all nucleic acid species. While modifications in the tRNA anticodon loop directly contribute to translation-related functions in the ribosome, the functions of tRNA body modifications are less known. One example of such a modification is dihydrouridine (D) - the second most abundant tRNA modification across biology. D is found at multiple positions in D loop that is important for tRNA tertiary structure and has been implicated in maintaining cellular levels of specific tRNAs. Nonetheless, no specific role has been identified for this modification in protein synthesis or cellular homeostasis. Adding to this mystery, dihydrouridine synthases (DUS) enzymes are dispensable for Saccharomyces cerevisiae growth under normal laboratory conditions. We hypothesized that D on tRNA has specific translation-related function in cells under unique stress conditions, as has been shown for several other tRNA modifications. To understand the molecular level function of D, a S. cerevisiae growth screen was used to test if the lack of DUS enzymes affects cell survival under stress conditions such as alternative carbon sources, metal ions, ribosome inhibition, oxidative stresses and temperature. While DUS mutants grew like WT cells under most conditions, DUS 1 and 4 mutants displayed severe growth phenotypes in the presence of translation inhibitors, hygromycin B and cycloheximide. Translation experiments using an E. coli reconstituted in vitro system suggests that tRNAPhe purified from E. coli dusC cells has an improved translocation rate in the presence of hygromycin B. Both in vitro and S. cerevisiae in vivo data suggest that DUS mutants promote frameshifting events. Taken together, our findings indicate that D modification in tRNA is involved in fine-tuning translation speed, particularly at the translocation step in protein synthesis, while maintaining the correct reading frame.
Keywords: tRNA modifications, protein synthesis, dihydrouridine