Talk abstracts
Talk on Friday 04:00-04:15pm submitted by Meredith Purchal
Pseudouridine synthase 7 is an opportunistic enzyme that binds and modifies substrates with diverse sequences and structures
Meredith K. Purchal (Program in Chemical Biology, University of Michigan), Daniel E. Eyler, Mehmet Tardu, Megan M. Korn (Department of Chemistry, University of Michigan), Monika K. Franco, Taslima Khan (Program in Chemical Biology, University of Michigan), Ryan McNassor, Hari Sharma, Leena Malik (Department of Chemistry, University of Michigan), Markos Koutmos (Program in Chemical Biology, Department of Chemistry, Department of Biophysics, University of Michigan), Kristin S. Koutmou (Program in Chemical Biology, Department of Chemistry, University of Michigan)
Abstract:
Pseudouridine is a ubiquitous RNA modification incorporated by pseudouridine synthase (Pus) enzymes into hundreds of non-coding and protein coding RNA substrates. Here, we determined the contributions of substrate structure and protein sequence to binding and catalysis by pseudouridine synthase 7 (Pus7), one of the principal mRNA modifying Pus enzymes. Pus7 catalyzes pseudouridine formation into a diverse set of mRNA sequences and is distinct among the Pus proteins because it shares minimal sequence homology with other pseudouridine synthase family members. We solved the first crystal structure of Saccharomyces cerevisiae Pus7, revealing the architecture of the eukaryotic specific insertion domains thought to be responsible for the expanded substrate scope of Pus7. Additionally, we identified amino acids and an insertion in the protein important for substrate binding and modification. Our data demonstrate that Pus7 preferentially binds substrates > 25 nucleotides in length possessing the previously identified UGUAR (R = purine) consensus sequence. Furthermore, we observed that RNA secondary structure is not a strong requirement for Pus7 binding. In contrast, the rate constants for pseudouridine incorporation are more influenced by RNA structure, with Pus7 modifying UGUAR sequences in less structured contexts more quickly. Although less structured substrates were preferred, Pus7 fully modified every tRNA, mRNA and non-natural RNAs containing the consensus recognition sequence we tested. These data suggest that Pus7 is a promiscuous enzyme, and lead us to propose a model for Pus7 substrate selection wherein the enzyme targets its substrates largely based upon the accessibility of a modifiable sequence.
Keywords: RNA modifications, Pseudouridine synthase, crystallography