Systemic insight into bacterial protein processing by live-cell single-molecule tracking

A single Escherichia coli cell contains thousands of different kinds of proteins in a total of millions of copies, all working together to ensure that the cell can grow and divide. New proteins are made by large cellular machines known as ribosomes, and during optimal growth, the ribosomes must produce roughly 20 new proteins every second. To ensure that these new proteins end up correctly folded at their intended cellular location with correct chemical modifications, the ribosome and the nascent protein require assistance from a number of processing factors. Importantly, different proteins require help from different factors. How these factors find their target proteins among the tens of thousands of ribosomes, each translating one of thousands of possible proteins, and engage with them in a coordinated fashion without impeding the other factors, remains mysterious. Traditional in vitro studies have provided high-resolution details about the function of individual factors. However, these fail to mimic the dynamic, crowded conditions of a living cell required to get the full picture of the system. In this work, we have employed a range of single-molecule super-resolution microscopy techniques to study different aspects of protein synthesis directly inside living E. coli cells. In Paper I and Paper II, we used two different microscopy methods to benchmark the cellular activity of Trigger factor (TF), a processing factor that guides the folding of new proteins. We discovered that TF binding to ribosomes is more dynamic than previously perceived. We also saw that TF competes for ribosome binding with another vital factor, namely, the Signal recognition particle. In Paper III, we took a closer look on the biogenesis of outer-membrane proteins, assessing the possibility of using in vivo single-molecule FRET as a reporter for their folding. In all, this work provides fundamental insight into how proteins are processed within live bacterial cells. Such knowledge is key to developing new treatments for bacterial infections and for understanding human diseases linked to protein misfolding.