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 #
5R01GM099687-04
Application #
8727064
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Lewis, Catherine D
Project Start
2011-09-30
Project End
2016-08-31
Budget Start
2014-09-01
Budget End
2015-08-31
Support Year
4
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Stanford University
Department
Biology
Type
Schools of Medicine
DUNS #
City
Stanford
State
CA
Country
United States
Zip Code
94304
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 163:1267-80
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
Sagan, Selena M; Chahal, Jasmin; Sarnow, Peter (2015) cis-Acting RNA elements in the hepatitis C virus RNA genome. Virus Res 206:90-8
Capece, Mark C; Kornberg, Guy L; Petrov, Alexey et al. (2015) A simple real-time assay for in vitro translation. RNA 21:296-305
Chen, Jin; Petrov, Alexey; Johansson, Magnus et al. (2014) Dynamic pathways of -1 translational frameshifting. Nature 512:328-32
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

Showing the most recent 10 out of 20 publications