Investigating the Determinants of Ribosome Pausing in Bacteria and Cellular Function of Trigger Factor

Biological systems use proteins to drive just about every cellular process. Thus one would gain much insight into the physiology of an organism by knowing the catalog of proteins that are being actively expressed. Ribosome profiling (deep sequencing of ribosome-protected mRNA footprints) enables one to monitor protein production genome-wide. Here, I describe the application of ribosome profiling to the Gram-negative bacterium Escherichia coli, and show that translation control is pervasive. In addition, I review the regulatory mechanisms that govern transient ribosome pausing, which can influence a number of cotranslational processes, such as folding and localization of the nascent chain. I continue this discussion by showing that pausing is widespread, and mainly stems from transient interactions made between the anti-Shine-Dalgarno sequence of the 16S ribosomal RNA and the messenger RNA template, rather than the cellular concentrations of transfer RNA. Finally, I describe a new application of the ribosome profiling approach that enables one to monitor cotranslational processes in vivo. Here, I characterize the cotranslational action of the molecular chaperone trigger factor. This study unexpectedly revealed that trigger factor does not engage nascent chains until well after synthesis, contrary to in vitro findings, and that outer membrane porin proteins are the most prominent substrates of this chaperone.