Project Summary Transfer RNAs (tRNAs) are central to translation of the genetic code to amino acid building blocks during protein synthesis on the ribosome. The human genome encodes 417 tRNA genes (gtrnadb.ucsc.edu), more than what is needed to translate the 61 sense codons. The diversity of tRNA genes in the human genome is previously unanticipated. We do not yet know which tRNA genes support protein synthesis and how we can image their activity and dynamics. While there is a strong need for robust labeling and imaging of tRNAs in live cells, progress has been slow. Without the convenience of making genetic fusions, such as protein fusions with fluorescent tags (GFP, YFP, etc), the current technology of tRNA labeling is limited to ex vivo conjugation with a fluorophore, followed by transfection or electroporation of the labeled tRNA into a cell. The disadvantage of the ex vivo approach is that the labeled tRNA is not synchronized with cell division. We were the first to develop a genetic fusion technology of tRNA with an RNA aptamer in an approach that is entirely based on nucleic acid replication to express and monitor tRNA for live-cell imaging. We have shown that an E. coli tRNA fused with a ?Spinach? aptamer emits spinach-like fluorescence when expressed in E. coli. We have further shown that this Spinach- tRNA is accommodated by the E. coli endogenous protein synthesis machinery, including amino-acid charging by an aminoacyl-tRNA synthetase, access to the ribosome by translation factors, and interaction with the ribosome to make a peptide bond at both the A (aminoacyl-tRNA)- and P (peptidyl-tRNA)-site. The success of the Spinach-tRNA technology was unexpected, given that both the tRNA and the aptamer are of a similar size and that each has a well-defined tertiary structure. We propose to bring this technology to human cells and explore additional aptamers, such as ?Mango? that emits a mango-like color. In Aim 1, we will use our genome- wide screening platform to identify tRNA genes that support protein synthesis by the ability to suppress a pre- mature termination codon in a reporter gene. All of the 417 tRNA genes will be screened for suppression at all three stop codons (UAG, UGA, and UAA) to identify the subset that are active in protein synthesis as tools for genome research. In Aim 2, we will perform another genome-wide screen to identify tRNAs that can be fused with an aptamer for live-cell imaging. We will generate a Spinach- and a Mango-library and screen for fusions in each that are active for protein synthesis. This will allow us to pair a Spinach- with a Mango-tRNA in a novel design that monitors FRET (Foster resonance energy transfer) when they occupy adjacent sites on the same ribosome during the making of a nascent peptide bond. By using FRET to focus on tRNAs in association with ribosomes, rather than those non-associated, we will quantify levels of protein synthesis in response to drug treatment and determine how protein synthesis may oscillate in the progression of a cell cycle. This project is at the forefront of powerful developments of new technologies for live-cell imaging of tRNA in the human genome.