Translation is the endpoint of gene expression. Messenger RNAs are decoded using a complex machinery that has the ribosome as its centerpiece. Despite its importance, methods to track translation of cellular mRNAs remain undeveloped. Here, we build on years of method development in single-molecule translation to propose a novel, real-time method to track translation at codon resolution. Our approach uses fluorescently labeled tRNAs, ribosomes and other ligands to map translation start sites, coding sequences and termination sites. We build on our preliminary data demonstrating that real time single-molecule analysis of translation can be performed using fluorescently labeled tRNAs and ribosomes, harnessing recently-developed DNA sequencing instrumentation. We will focus our development efforts during the proposed funding period on three specific aims that will (1) create a collection of benchmarked fluorescent reagents for single-molecule translational profiling (2) use these reagents to characterize translational start sites, reading frames, termination sites and drug effects in three organisms: E. coli, yeast and human and (3) characterize rare translational phenomena, such as frameshifting, in these organisms. The goal of this work is provide real-time, genome-wide analysis of translational processes in these organisms. The endpoint of this proposal will be a core research facility that is dedicated to single-molecule translational analysis and providing access to the broader biomedical community. We believe these results will lead to a deeper understanding of the role of translational regulation in human disease.
| Choi, Junhong; Indrisiunaite, Gabriele; DeMirci, Hasan et al. (2018) 2'-O-methylation in mRNA disrupts tRNA decoding during translation elongation. Nat Struct Mol Biol 25:208-216 |
| Prabhakar, Arjun; Capece, Mark C; Petrov, Alexey et al. (2017) Post-termination Ribosome Intermediate Acts as the Gateway to Ribosome Recycling. Cell Rep 20:161-172 |
| Navon, Sharon Penias; Kornberg, Guy; Chen, Jin et al. (2016) Amino acid sequence repertoire of the bacterial proteome and the occurrence of untranslatable sequences. Proc Natl Acad Sci U S A 113:7166-70 |
| Choi, Junhong; Ieong, Ka-Weng; Demirci, Hasan et al. (2016) N(6)-methyladenosine in mRNA disrupts tRNA selection and translation-elongation dynamics. Nat Struct Mol Biol 23:110-5 |
| Petrov, Alexey; Grosely, Rosslyn; Chen, Jin et al. (2016) Multiple Parallel Pathways of Translation Initiation on the CrPV IRES. Mol Cell 62:92-103 |
| Chen, Jin; Coakley, Arthur; O'Connor, Michelle et al. (2015) Coupling of mRNA Structure Rearrangement to Ribosome Movement during Bypassing of Non-coding Regions. Cell 163:1267-1280 |
| Nilsson, Ola B; Hedman, Rickard; Marino, Jacopo et al. (2015) Cotranslational Protein Folding inside the Ribosome Exit Tunnel. Cell Rep 12:1533-40 |
| Sagan, Selena M; Chahal, Jasmin; Sarnow, Peter (2015) cis-Acting RNA elements in the hepatitis C virus RNA genome. Virus Res 206:90-8 |
| Fuchs, Gabriele; Petrov, Alexey N; Marceau, Caleb D et al. (2015) Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site. Proc Natl Acad Sci U S A 112:319-25 |
| Capece, Mark C; Kornberg, Guy L; Petrov, Alexey et al. (2015) A simple real-time assay for in vitro translation. RNA 21:296-305 |
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