Project Summary/Abstract Rationale: The speed of protein synthesis can impact all co-translational processes, from folding to degradation of the nascent chain. It was not until 3 years ago that we had the first global views of the speed of translation with sub-codon resolution in vivo. The enabling technology is ribosome profiling?deep sequencing of ribosome-protected mRNA fragments?developed in the Weissman lab at UCSF. By combining ribosome profiling with computational approaches, I have now initiated an effort to decipher how translational pausing regulates protein synthesis. Since starting at UCSF, I made the surprising discovery that the majority of translational pause sites in bacteria occur at internal Shine-Dalgarno (SD) sequences, driven by their interaction with the anti-Shine- Dalgarno (antiSD) region of the elongating ribosome. The current paradigm, established by Shine and Dalgarno in 1975, is that the main role of the ribosomal antiSD region is to define translation initiation sites in prokaryotes. My finding that there is conserved and ubiquitous pausing at internal SD sites suggests a distinct function for the antiSD region during the elongation phase of translation. In fact, recent genome sequencing data have revealed that, although the antiSD region of ribosomal RNA is highly conserved throughout prokaryotes, many bacterial and archaeal species do not use it for translation initiation. Intriguingly, several intragenic SD sites are conserved across many species. I hypothesize that this novel function of antiSD during translational elongation is an important factor driving the conservation of the antiSD region. Objective: To understand the widespread use of SD-induced pausing, I propose to investigate the co- translational processes that are controlled by pausing sites identified by our genome-wide measurements. My immediate goals are to define the broader role of anti-Shine-Dalgarno sequence in prokaryotic translation, and to determine the role of translational pausing in protein folding, membrane insertion, and post-transcriptional regulation. This work will elucidate the principles governing the interplay between translational pausing and co- translational processes in all organisms including eukaryotes, which also exhibit ubiquitous, albeit mechanistically distinct pauses with unexplored functions. Coming from a background in physics, I am seeking to complement my analytical and optical skills with solid hands-on training in cell biology and biochemistry. In pursuit of these aims with my mentors' expertise in protein folding and stress responses, I will acquire both the knowledge base and a unique perspective to launch my own independent investigation on gene expression and protein synthesis from the mechanistic level to the systems level.