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.

National Institute of Health (NIH)
Research Project (R01)
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Macromolecular Structure and Function C Study Section (MSFC)
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Lewis, Catherine D
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Stanford University
Schools of Medicine
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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; 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
Simonetti, Angelita; Marzi, Stefano; Billas, Isabelle M L et al. (2013) Involvement of protein IF2 N domain in ribosomal subunit joining revealed from architecture and function of the full-length initiation factor. Proc Natl Acad Sci U S A 110:15656-61
Tsai, Albert; Uemura, Sotaro; Johansson, Magnus et al. (2013) The impact of aminoglycosides on the dynamics of translation elongation. Cell Rep 3:497-508
Chen, Jin; Tsai, Albert; Petrov, Alexey et al. (2012) Nonfluorescent quenchers to correlate single-molecule conformational and compositional dynamics. J Am Chem Soc 134:5734-7
Masuda, Tomoaki; Petrov, Alexey N; Iizuka, Ryo et al. (2012) Initiation factor 2, tRNA, and 50S subunits cooperatively stabilize mRNAs on the ribosome during initiation. Proc Natl Acad Sci U S A 109:4881-5

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