Translation of proteins by the ribosome is key aspect of life. Structural studies have provided a three- dimensional view of the 2.5 megaDalton 70S ribosomal particle, and revealed the active sites for decoding of genetic information and peptide bond formation. Mechanistic views have revealed the basic steps of translation: initiation, elongation and termination. However, limited dynamic information about how the translational machinery changes conformation and composition are available. Our research focuses on providing a dynamic view of translation using single-molecule approaches. The current proposal builds on the progress in observing ribosomal dynamics in real time using single- molecule fluorescence approaches. We will monitor the underlying dynamics of translation in four specific aims. First, we will explore the mechanism of initiation, in which the 70S ribosomal particle assembles at the correct start codon;we will use single molecule fluorescence to understand intersubunit joining and dynamics, the role of factors in initiation, and movements of tRNAs, factors and ligands during the process. These experiments will be complemented by single-molecule force measurements to map the energetics of mRNA-ribosome interaction.
In aim 2, we will explore the nature of intersubunit conformational changes that underlie both initiation and elongation. We will determine the nature of the barrier to rotation using both fluorescence and force and explore how factor and tRNA dynamics are correlated to ribosomal motions. We will determine the effects of ribosomal mutations and composition, as well as antibiotics, on conformational dynamics.
In aim 3, we will apply new single-molecule instrumentation to perform single-molecule experiments under high concentrations (50nM-5M) of labeled tRNAs, ribosomes or factors. These experiments harness zero mode waveguides, and will allow us to observe rapid changes in ribosome composition, and correlate conformational changes with each other. Finally, in aim 4, the dynamics of ribosomal translation will be explored during translation to understand codon and mRNA context effects. We will use intersubunit FRET, tRNA colocalization and ribosome mRNA -FRET to determine dynamics effects on model mRNAs, and then use these systems to explore the dynamics of rare recoding events. The results of this project should reveal a dynamic view of translation and continue to provide insights into the mechanism of action of antibiotics.

Public Health Relevance

The ribosome synthesizes proteins in the cell. This proposal aims to provide a molecular movie of how the ribosome performs this task, and how synthesis of proteins might be controlled in the cell. We will use methods of single-molecule biophysics to explore ribosome dynamics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM051266-20
Application #
8470171
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Lewis, Catherine D
Project Start
1995-05-01
Project End
2014-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
20
Fiscal Year
2013
Total Cost
$334,552
Indirect Cost
$123,944
Name
Stanford University
Department
Biology
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
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
Choi, Junhong; Puglisi, Joseph D (2017) Three tRNAs on the ribosome slow translation elongation. Proc Natl Acad Sci U S A 114:13691-13696
Prabhakar, Arjun; Choi, Junhong; Wang, Jinfan et al. (2017) Dynamic basis of fidelity and speed in translation: Coordinated multistep mechanisms of elongation and termination. Protein Sci 26:1352-1362
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
Sierra, Raymond G; Gati, Cornelius; Laksmono, Hartawan et al. (2016) Concentric-flow electrokinetic injector enables serial crystallography of ribosome and photosystem II. Nat Methods 13:59-62
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
Puglisi, Joseph D (2015) Synthetic biology: Ribosomal ties that bind. Nature 524:45-6
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

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