Protein synthesis is a highly conserved process in all kingdoms of life that can be broken down into four distinct phases: initiation, elongation, termination and ribosome recycling. The broad goals of this research program are to use biochemical and genomic approaches to shed light on the common and distinctive molecular features of translation elongation, termination, and recycling in bacteria and eukaryotes, and their control. Here we are particularly focused on one aspect of translational control in which ribosomal stalling triggers a cellular response leading to mRNA decay, targeted proteolysis, and ribosome recycling. In particular, we focus on a highly conserved stalling motif, the poly-basic peptide sequence that is of particular relevance in eukaryotic cells where alternative polyadenylation site usage commonly leads to """"""""non-stop"""""""" mRNAs. We will use in vitro biochemistry and in vivo ribosome profiling to look at the molecular mechanics of this biologically important and conserved process. More specifically, we propose (1) to use reporters and our previously established in vitro reconstituted translation systems (with E. coli and S. cerevisiae components) to ask a series of questions about how poly-basic sequences disrupt ribosome function during elongation, (2) to use these same reporters and in vitro biochemistry to define how different extra-ribosomal factors engage the ribosome and impact elongation, termination and recycling and (3) to use recently developed ribosome profiling approaches to define the biologically relevant in vivo targets, their molecular features, and the factors that respond in the cell to resolve the crisis. We anticipate that the synergy of these approaches will be powerful in defining biologically relevant mechanism.
mRNA surveillance pathways (including non-stop decay, NSD) are critical in regulating gene expression throughout the three kingdoms of life. Of key relevance to this proposal, non-stop mRNAs are commonly generated (as much as 5-10% of transcripts) in eukaryotic cells when cryptic poly(A) sites are utilized;such events have been implicated as relevant for a number of diseases [Klauer, 2012 #100]. Mechanistic understanding of NSD will be required for the development of effective therapies to treat such diseases.
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