Title: Genome-wide Mapping of Ribosome Occupancies with Inhibitor-Induced mRNA Covalent Labeling Ribosome profiling is a powerful method to study translation through genome-wide mapping of translating ribosomes. This breakthrough technology has illuminated many facets of protein synthesis, such as ribosomal pausing, alternative initiation sites, and protein synthesis rates across numerous cellular systems. Although recent innovations have reduced ribosome profiling to single cells, ribosome profiling typically requires large numbers of cells to conduct and therefore is not routinely used to study rare cell types and clinical samples. Furthermore, ribosome profiling fragments mRNA and hence cannot map multiple ribosomes along a single mRNA molecule and requires integration of separate mRNA sequencing datasets to account for the total number of transcripts. New efficient methods to simultaneously measure ribosome occupancies and mRNA abundance by preserving mRNA integrity would further enable the global study of translational regulation and provide new fundamental insight into translation dynamics. To bridge these technological gaps, here we propose to develop new small molecule tools based off the natural product ribosome inhibitors to chemically footprint ribosome positioning atop mRNA transcripts through covalent labeling. Our central hypothesis is that these mRNA chemical footprints can be detected through error prone reverse transcription and subsequent mRNA sequencing to map ribosome occupancies while maintaining the integrity of the mRNA. This approach is termed ribosome mutational profiling (Ribo-MaP). To develop Ribo-MaP, the first aim proposes the structure-based design of natural product derivatives that potently inhibit ribosome elongation while maintaining polysome stability. Critically, these derivatives are based off emetine, a ribosome inhibitor which contacts the mRNA template to block translation. Furthermore, photoaffinity labeling groups will be attached to these emetine derivatives and the ability to covalently label mRNA will be tested. Additionally, an alternative approach for mRNA covalent labeling is proposed, in which emetine derivatives will be functionalized with reactive acylating groups to label the proximal mRNA 2'- hydroxyl. In both approaches, mRNA labeling efficiency and the ability to readout covalent modifications as mutations in error prone reverse transcription will be measured. In the second aim, the ability of Ribo-MaP to map ribosome occupancies genome-wide will be evaluated and benchmarked against conventional ribosome profiling. We expect that Ribo-MaP will bypass inefficient steps in ribosome profiling and drop into the streamlined workflow of both robust mRNA sequencing methods and new emerging technologies for single-cell and long-read mRNA sequencing. Altogether, Ribo-MaP would provide a highly enabling method to broadly study protein synthesis and ribosome dynamics in cells.