The Buskirk group studies protein synthesis with a focus on ribosome stalling and the mechanisms that cells use to restart or recycle stalled ribosomes. In 2014 the group moved to Hopkins to the lab of our long‐term collaborator Rachel Green, where we continue to pursue these projects with independent NIH funding.
Pauses in translation occur when the ribosome encounters strong secondary structures or clusters of rare codons in mRNA transcripts. Pauses can also be induced by interactions of the nascent polypeptide with the exit tunnel and active site. Through genetic screens, we identified nascent peptide sequences that block peptidyl transfer and peptide release (Woolstenhulme 2013). The fact that many of these motifs pause ribosomes at consecutive Pro codons helped to establish that Pro acts as both a poor peptidyl donor and a poor peptidyl acceptor. We are interested in the mechanism of how consecutive Pro residues alter the active site geometry within the ribosome to inhibit peptidyl transfer as well as how elongation factor P (EFP) counteracts this effect, alleviating pausing at polyproline motifs.
When ribosomes stall for prolonged periods of time, for example at the 3’‐end of mRNAs lacking a stop codon, they are rescued and recycled by the conserved tmRNA‐SmpB complex or by ArfA/B in certain species. These factors must distinguish stalled ribosomes from actively translating ones to avoid aborting productive protein synthesis. We continue to study how this selectivity is achieved using genetic methods and pre‐steady state kinetics (Miller 2014).
Variations in elongation rates have been implicated in gene regulation, mRNA decay, and protein folding. We are working with Patricia Clark and the Protein Translation Research Network to further characterize the effect of elongation rate on protein folding. The ribosome profiling method developed by Ingolia and Weissman may provide a means to further flesh out promising links between translational pauses and important biological outcomes. We have improved the resolution of bacterial ribosome profiling data and analyzed the effects of pausing in cells lacking EFP (Woolstenhulme 2015), and we continue to develop and employ this technique to identify and characterize translational pauses.Recent Publications