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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM099687-02
Application #
8338860
Study Section
Special Emphasis Panel (ZRG1-BCMB-A (51))
Program Officer
Lewis, Catherine D
Project Start
2011-09-30
Project End
2016-08-31
Budget Start
2012-09-01
Budget End
2013-08-31
Support Year
2
Fiscal Year
2012
Total Cost
$721,972
Indirect Cost
$262,117
Name
Stanford University
Department
Biology
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Chen, Jin; Petrov, Alexey; Johansson, Magnus et al. (2014) Dynamic pathways of -1 translational frameshifting. Nature 512:328-32
Tsai, Albert; Kornberg, Guy; Johansson, Magnus et al. (2014) The dynamics of SecM-induced translational stalling. Cell Rep 7:1521-33
Noriega, Thomas R; Tsai, Albert; Elvekrog, Margaret M et al. (2014) Signal recognition particle-ribosome binding is sensitive to nascent chain length. J Biol Chem 289:19294-305
Chen, Jin; Dalal, Ravindra V; Petrov, Alexey N et al. (2014) High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence. Proc Natl Acad Sci U S A 111:664-9
Noriega, Thomas R; Chen, Jin; Walter, Peter et al. (2014) Real-time observation of signal recognition particle binding to actively translating ribosomes. Elife 3:
Johansson, Magnus; Chen, Jin; Tsai, Albert et al. (2014) Sequence-dependent elongation dynamics on macrolide-bound ribosomes. Cell Rep 7:1534-46
Chen, Jin; Petrov, Alexey; Tsai, Albert et al. (2013) Coordinated conformational and compositional dynamics drive ribosome translocation. Nat Struct Mol Biol 20:718-27
O'Leary, Seán E; Petrov, Alexey; Chen, Jin et al. (2013) Dynamic recognition of the mRNA cap by Saccharomyces cerevisiae eIF4E. Structure 21:2197-207