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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.

PART IVertebrate syndapin (Synd) was identified as a dynamin interacting protein, which binds Wiskott-Aldrich Syndrome protein (WASp). WASp is a known activator of the Arp-2/3 complex (actin related protein) that promotes actin polymerization. In Drosophila, Synd has a postsynaptic distribution at neuromuscular junctions (NMJ). To determine the synaptic function of Synd, we have generated and characterized Drosophila Synd loss-of-function mutants. These mutants display a NMJ overgrowth phenotype manifested by increased synaptic span, bouton number, and extent of branching. The synd mutants also contain hyperbranching boutons that give rise to multiple “satellite” boutons. We can rescue this phenotype by expression of a synd transgene postsynaptically, but not presynaptically. Loss of synd resembles the NMJ morphological phenotype of wsp loss-of-function mutants, suggesting a common functional pathway. Furthermore, the NMJ overgrowth phenotype is most pronounced in synd and wsp double mutants and dependent on gene dosage – demonstrating that the two genes interact. Finally, relative postsynaptic staining of Wsp is decreased in a synd mutant, but Synd remains intact in a wsp mutant. Taken together, this suggests that Syndapin serves as an adaptor protein recruiting Wsp postsynaptically to the NMJ. Loss of synd is likely to modulate synapse growth by disrupting Wsp localization and function. PART IIThe 5-HT1A receptor is a metabotropic G protein-coupled receptor (GPCR) linked to the Gi/o signaling pathway and has been specifically implicated in the pathogenesis of depression and anxiety. To understand and precisely control 5-HT1A signaling, we created a light-activated GPCR, which targets into 5-HT1A receptor domains and substitutes for endogenous 5-HT1A receptors. To induce 5-HT1A-like targeting, vertebrate rhodopsin (Rh) was tagged with the C-terminal domain (CT) of 5-HT1A (Rh-CT5-HT1A). Rh-CT5-HT1A activates G protein-coupled inward rectifying K+ channels (GIRK) in response to light and causes membrane hyperpolarization in hippocampal neurons, similar to the agonist-induced responses of the 5-HT1A receptor. The intracellular distribution of Rh-CT5-HT1A resembles that of wild type 5-HT1A receptor; Rh-CT5-HT1A localizes somatodendritically and efficiently traffics to distal dendritic processes. Additionally, neuronal expression of Rh-CT5-HT1A, but not Rh, inhibits responses to 5-HT1A agonist, suggesting that Rh-CT5-HT1A and endogenous 5-HT1A receptors compete to interact with the same trafficking machinery. Finally, Rh-CT5-HT1A is able to substitute for absent 5-HT1A signaling in cultured neurons and dorsal raphe slices of KO mice, showing that Rh-CT5-HT1A functionally compensates for native 5-HT1A. Thus, as an optogenetic tool, Rh-CT5-HT1A has the potential to directly correlate in vivo 5-HT1A signaling with 5-HT neuron activity and behavior in both wild type animals and animal models of neuropsychiatric disease.