By using single-molecule fluorescence resonance energy transfer (smFRET), we observe the real-time dynamic coupling between the ribosome, labeled at the L1 stalk, and transfer RNA (tRNA). We find that an interaction between the ribosomal L1 stalk and the newly deacylated tRNA is established spontaneously upon peptide bond formation; this event involves coupled movements of the L1 stalk and tRNAs as well as ratcheting of the ribosome. In the absence of elongation factor G, the entire pretranslocation ribosome fluctuates between just two states: a nonratcheted state, with tRNAs in their classical configuration and no L1 stalk-tRNA interaction, and a ratcheted state, with tRNAs in an intermediate hybrid configuration and a direct L1 stalk-tRNA interaction. We demonstrate that binding of EF-G shifts the equilibrium toward the ratcheted state. Real-time smFRET experiments reveal that the L1 stalk-tRNA interaction persists throughout the translocation reaction, suggesting that the L1 stalk acts to direct tRNA movements during translocation.
机构:Univ Calif Santa Cruz, Dept Mol Cell & Dev Biol, Ctr Mol Biol RNA, Santa Cruz, CA 95064 USA
Ermolenko, Dmitri N.
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Majumdar, Zigurts K.
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机构:Univ Calif Santa Cruz, Dept Mol Cell & Dev Biol, Ctr Mol Biol RNA, Santa Cruz, CA 95064 USA
Majumdar, Zigurts K.
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Hickerson, Robyn P.
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机构:Univ Calif Santa Cruz, Dept Mol Cell & Dev Biol, Ctr Mol Biol RNA, Santa Cruz, CA 95064 USA
Hickerson, Robyn P.
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Spiegel, P. Clint
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机构:Univ Calif Santa Cruz, Dept Mol Cell & Dev Biol, Ctr Mol Biol RNA, Santa Cruz, CA 95064 USA
Spiegel, P. Clint
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Clegg, Robert M.
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机构:Univ Calif Santa Cruz, Dept Mol Cell & Dev Biol, Ctr Mol Biol RNA, Santa Cruz, CA 95064 USA
Clegg, Robert M.
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Noller, Harry F.
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Univ Calif Santa Cruz, Dept Mol Cell & Dev Biol, Ctr Mol Biol RNA, Santa Cruz, CA 95064 USAUniv Calif Santa Cruz, Dept Mol Cell & Dev Biol, Ctr Mol Biol RNA, Santa Cruz, CA 95064 USA
机构:Univ Calif Santa Cruz, Dept Mol Cell & Dev Biol, Ctr Mol Biol RNA, Santa Cruz, CA 95064 USA
Ermolenko, Dmitri N.
;
Majumdar, Zigurts K.
论文数: 0引用数: 0
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机构:Univ Calif Santa Cruz, Dept Mol Cell & Dev Biol, Ctr Mol Biol RNA, Santa Cruz, CA 95064 USA
Majumdar, Zigurts K.
;
Hickerson, Robyn P.
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h-index: 0
机构:Univ Calif Santa Cruz, Dept Mol Cell & Dev Biol, Ctr Mol Biol RNA, Santa Cruz, CA 95064 USA
Hickerson, Robyn P.
;
Spiegel, P. Clint
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h-index: 0
机构:Univ Calif Santa Cruz, Dept Mol Cell & Dev Biol, Ctr Mol Biol RNA, Santa Cruz, CA 95064 USA
Spiegel, P. Clint
;
Clegg, Robert M.
论文数: 0引用数: 0
h-index: 0
机构:Univ Calif Santa Cruz, Dept Mol Cell & Dev Biol, Ctr Mol Biol RNA, Santa Cruz, CA 95064 USA
Clegg, Robert M.
;
Noller, Harry F.
论文数: 0引用数: 0
h-index: 0
机构:
Univ Calif Santa Cruz, Dept Mol Cell & Dev Biol, Ctr Mol Biol RNA, Santa Cruz, CA 95064 USAUniv Calif Santa Cruz, Dept Mol Cell & Dev Biol, Ctr Mol Biol RNA, Santa Cruz, CA 95064 USA