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Heterogeneous pathways and timing of factor departure during translation initiation

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Abstract

The initiation of translation establishes the reading frame for protein synthesis and is a key point of regulation1. Initiation involves factor-driven assembly at a start codon of a messenger RNA of an elongation-competent 70S ribosomal particle (in bacteria) from separated 30S and 50S subunits and initiator transfer RNA. Here we establish in Escherichia coli, using direct single-molecule tracking, the timing of initiator tRNA, initiation factor 2 (IF2; encoded by infB) and 50S subunit joining during initiation. Our results show multiple pathways to initiation, with orders of arrival of tRNA and IF2 dependent on factor concentration and composition. IF2 accelerates 50S subunit joining and stabilizes the assembled 70S complex. Transition to elongation is gated by the departure of IF2 after GTP hydrolysis, allowing efficient arrival of elongator tRNAs to the second codon presented in the aminoacyl-tRNA binding site (A site). These experiments highlight the power of single-molecule approaches to delineate mechanisms in complex multicomponent systems.

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Figure 1: Pathways leading to 30S PIC formation.
Figure 2: 50S subunit joining to 30S PIC.
Figure 3: Timing of IF2 departure and elongator tRNA arrival after 70S complex formation.
Figure 4: The heterogeneous pathways of translation initiation.

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Acknowledgements

Supported by National Institutes of Health grant GM51266 (J.D.P.) and the Japan Science and Technology Agency (S.U.).

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Authors and Affiliations

  1. Department of Structural Biology, Stanford University School of Medicine, Stanford, 94305-5126, California, USA

    Albert Tsai, Alexey Petrov, R. Andrew Marshall, Sotaro Uemura & Joseph D. Puglisi

  2. Department of Applied Physics, Stanford University, Stanford, 94305-4090, California, USA

    Albert Tsai

  3. McKinsey & Company - Silicon Valley, 3705A Hansen Way, Palo Alto, 94304, California, USA

    R. Andrew Marshall

  4. Pacific Biosciences, 1380 Willow Rd, Menlo Park, 94025, California, USA

    Jonas Korlach

  5. Omics Science Center, RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama City, Kanagawa 230-0045, Japan,

    Sotaro Uemura

  6. Stanford Magnetic Resonance Laboratory, Stanford University School of Medicine, Stanford, 94305-5126, California, USA

    Joseph D. Puglisi

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  1. Albert Tsai
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  2. Alexey Petrov
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  4. Jonas Korlach
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  5. Sotaro Uemura
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  6. Joseph D. Puglisi
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Contributions

A.T., A.P. and S.U. conducted the experiments and performed the analysis; R.A.M. prepared and provided experimental materials; J.K. provided technical expertise with instrumentation and data processing; S.U. and J.D.P. designed experiments; and all authors discussed results and wrote the manuscript.

Corresponding authors

Correspondence to Sotaro Uemura or Joseph D. Puglisi.

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Tsai, A., Petrov, A., Marshall, R. et al. Heterogeneous pathways and timing of factor departure during translation initiation. Nature 487, 390–393 (2012). https://doi.org/10.1038/nature11172

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