Coordination of translation and assembly of protein complexes

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A large percent of any proteome of living cells acquires function only when participating in oligomeric protein complexes, but little is known about the process of protein complex assembly. In the previous funding period, we established that in five of the six bacterial protein complexes examined, complex assembly begins co-translationally involving subunit association with nascent chains of partner subunits once the contact interphase is exposed on the surface of the translating ribosome. We further found in proof-of-principle experiments using heterodimeric bacterial luciferase encoded by the luxA-luxB operon, that contiguous positioning of luxA-luxB on the polycistronic mRNA allows localized translation promoting efficient complex assembly in E. coli cells. This suggests a widespread mechanism which links protein complex assembly and function directly to genetically encoded and positional (physical) information. This conceptual advance now provides an area rich with regulatory potential which we propose to explore in the upcoming funding period.We will use a set of suitable protein complexes, and focus on mechanistic aspects of protein complex assembly in E. coli and how the assembly mechanism integrates with the actively translating ribosome. Specifically, we will first investigate nascent chain interactions per se. Using ribosome profiling, biochemical and genetic methodologies, we will investigate structural and kinetic features of nascent chain interactions with partner subunits (in collaboration with N. Budisa and M. Rodnina), and provide quantitative data for a mathematical description of an assembly reaction by our collaborator R. Lipowsky (Specific Aims I to III). We will then also investigate how organization of genes in operons affects co-translational assembly and determine the effect of translation speed on subunit assembly (Specific Aims IV and V). This will elucidate the interplay between physical and genetic information decoded and transmitted during translation into 3-dimensional protein complex assembly, and cellular function.