Protein synthesis by the ribosome is central to life and disease. Biochemical, kinetic and structural studies have provided detailed views of the intermediates and mechanisms in translation. To complement these approaches, we developed single-molecule fluorescence methods to track both conformational and compositional dynamics of the translation machinery in real time. Here we build on our methodological developments over the past funding period, and leverage the strengths of single-molecule methods watch translation in real time.
In Aim 1 we focus on basic steps of translation: the timing of initiation events, focusing on IF1 and IF3 and initiator tRNA, termination driven by RF1, 2 and 3 and recycling by RRF and EF-G. We will use conformational and compositional signals to track both simultaneously in real time using novel instrumentation.
In Aim 2, we will shift to investigate rar pausing, stalling and reading- frame changes that occur during elongation due to mRNA sequence or structure. We will study the role of SD interactions in reading frame, and the influence of RNA structure on elongation rates. We will then study the interplay of these RNA features that lead to -1 and +1 frame shifting and ribosome hopping, using single-molecule fluorescence to track pathways, branch points and mechanism.
In Aim 3, we examine factors that interact in the peptide exit tunnel to cause pausing and stalling, including ribosomal nascent chains and antibiotics such as macrolides. We will follow the evolution of stalling for several systems, including SecM, TnaC, and ErmCL, confirming the sequence features of the nascent chain required for stalling and their contributions to the kinetic pathway to the stalled or paused state. For ErmCL, we will determine the interplay of erythromycin and nascent chain that causes stalling, and correlate the stall behavior with drug occupancy. Finally, we will investigate generally the dynamics of macrolide binding to ribosomes, and how drug occupancy and dynamics change as a function of elongation for both sensitive and resistant ribosomes. The proposed research is buttressed by strong collaborations to support bulk kinetic analysis, reagent preparation, and in vivo correlation. The expected results should provide a dynamic overview of initiation, termination, and elongation, and how general dynamics are perturbed to change protein expression.

Public Health Relevance

Translation is inherently dynamic. Here we use single-molecule fluorescence to track conformational and compositional dynamics during initiation, elongation and termination, with an emphasis on rare events like stalling and frame shifting. We will observe translation directly in real time.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM051266-21
Application #
8698190
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Lewis, Catherine D
Project Start
1995-05-01
Project End
2018-04-30
Budget Start
2014-05-01
Budget End
2015-04-30
Support Year
21
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
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
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
Puglisi, Joseph D (2015) Protein synthesis. The delicate dance of translation and folding. Science 348:399-400
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
Nilsson, Ola B; Hedman, Rickard; Marino, Jacopo et al. (2015) Cotranslational Protein Folding inside the Ribosome Exit Tunnel. Cell Rep 12:1533-40
Puglisi, Joseph D (2015) Synthetic biology: Ribosomal ties that bind. Nature 524:45-6
Capece, Mark C; Kornberg, Guy L; Petrov, Alexey et al. (2015) A simple real-time assay for in vitro translation. RNA 21:296-305
Puglisi, Joseph D (2015) RNA dances to center stage. RNA 21:712-3
Chen, Jin; Petrov, Alexey; Johansson, Magnus et al. (2014) Dynamic pathways of -1 translational frameshifting. Nature 512:328-32

Showing the most recent 10 out of 54 publications