A leading cause of Cystic Fibrosis (CF) is premature termination codons (PTCs) in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Suppression of translation termination at PTCs?i.e. PTC readthrough?to restore full-length CFTR protein may be a treatment strategy. Yet, current PTC readthrough drug candidates for CF are toxic (e.g. aminoglycosides) or ineffective (e.g. ataluren). Efficacy of PTC readthrough depends on efficiency of translation termination at the PTC. Thus, manipulating the molecular mechanisms of CFTR PTC termination to lower efficiency may improve PTC readthrough efficacy. However, strategies for such manipulations are limited in the absence of a detailed understanding of translation termination on CFTR PTCs. In the current model for normal termination, eukaryotic Release Factors 1 and 3 form a complex (eRF1?eRF3) that releases a newly synthesized protein from the ribosome. eRF1 recognizes a tetra-nucleotide stop codon at the end of an open reading frame, and catalyzes peptidyl-tRNA hydrolysis. Poly-A binding protein (PABP), which binds at 3? ends of mRNA, recruits eRF3 and enhances termination efficiency. However, it remains unclear how PABP, eRF1, eRF3, and the tetra-nucleotide stop codon recognize the PTC to produce truncated CFTR protein. The goal of this proposal is to determine the biochemical and structural mechanism of translation termination. With guidance from Dr. Andrei Korostelev (expert in biochemical and structural mechanisms of translation), Dr. Allan Jacobson (expert in premature translation termination and PTC read-through), Dr. Phillip Zamore (RNA biochemist), Dr. Chen Xu (cryo-EM instrumentalist), and Dr. Nikolaus Grigorieff (expert in cryo-EM method development), release assays will be optimized to study the efficiency of translation termination mediated by eukaryotic release factors, and ensemble time-resolved (ENTIRE) cryo-EM will be used to capture structural intermediates of enzymatic reactions.
Aim 1 will use defined mammalian translation systems to measure the individual effects of stop codon context, eRF1?eRF3, and PABP on the termination efficiencies (kcat/KM) of CFTR PTCs and the true stop codon.
Aim 2 will visualize how the ribosome terminates on CFTR PTC G542X in its natural sequence context using ENTIRE cryo-EM. Collecting data at multiple time points will identify conformational changes and interactions between mRNA sequence, eRF1?eRF3, and PABP during termination. To reveal the termination mechanism on CFTR PTCs, structures and their progression intermediates will be compared with those recorded on the true CFTR stop codon. If successful, this study will reveal key molecular determinants of CFTR PTC termination, and may inform strategies to induce PTC readthrough for CF treatment.
Cystic Fibrosis is caused by gene mutations that produce a defective form of a protein important for lung, liver, pancreas, and digestive tract function. This proposal will visualize how cells recognize this gene defect to produce dysfunctional protein. Findings from these studies will inform the design of therapies that allow cells to ignore this gene defect to treat Cystic Fibrosis patients.
