Journal
RNA
Volume 17, Issue 12, Pages 2189-2200Publisher
COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
DOI: 10.1261/rna.029033.111
Keywords
translocation; peptidyl-tRNA; EF-G; ribosome; A-site finger; hybrid states; single-molecule FRET
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Funding
- National Institute of Health [1R01GM079238]
- Human Frontiers in Science Program [RGY0088]
- National Science Foundation [0644129]
- Div Of Molecular and Cellular Bioscience
- Direct For Biological Sciences [0747230] Funding Source: National Science Foundation
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Translocation, the directional movement of transfer RNA (tRNA) and messenger RNA (mRNA) substrates on the ribosome during protein synthesis, is regulated by dynamic processes intrinsic to the translating particle. Using single-molecule fluorescence resonance energy transfer (smFRET) imaging, in combination with site-directed mutagenesis of the ribosome and tRNA substrates, we show that peptidyl-tRNA within the aminoacyl site of the bacterial pretranslocation complex can adopt distinct hybrid tRNA configurations resulting from uncoupled motions of the 3'-CCA terminus and the tRNA body. As expected for an on-path translocation intermediate, the hybrid configuration where both the 3'-CCA end and body of peptidyl-tRNA have moved in the direction of translocation exhibits dramatically enhanced puromycin reactivity, an increase in the rate at which EF-G engages the ribosome, and accelerated rates of translocation. These findings provide compelling evidence that the substrate for EF-G catalyzed translocation is an intermediate wherein the bodies of both tRNA substrates adopt hybrid positions within the translating ribosome.
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