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High-Efficiency "-1" and "-2" Ribosomal Frameshiftings Revealed by Force Spectroscopy
Te-Wei Tsai1, Haopeng Yang1, Heng Yin1
1Department of Biology and Biochemistry, ‡Department of Chemistry, University of Houston , Houston, Texas 77204, United States.
ACS Chemical Biology
|April 25, 2017
Summary
Researchers observed efficient -1 and -2 ribosomal frameshifting on mRNA lacking secondary structures. This study provides the first experimental evidence of multiple frameshifting steps and reveals the crucial role of GTP hydrolysis by EF-G·GTP.
Area of Science:
- Molecular Biology
- Genetics
- Biophysics
Background:
- Ribosomal frameshifting is a known but rare biological process.
- Frameshifting motifs lacking secondary mRNA structures are poorly understood experimentally.
Purpose of the Study:
- To experimentally investigate frameshifting on a GA7G slippery mRNA sequence without secondary structures.
- To provide the first evidence of multiple frameshifting steps and rare -2 frameshifting.
- To elucidate the mechanistic roles of EF-G·GTP and in-frame tRNAs in frameshifting.
Main Methods:
- Force-induced remnant magnetization spectroscopy with unique probing schemes.
- Toe-printing assay.
- Protein sequencing.
Main Results:
- Demonstrated highly efficient -1 and -2 frameshifting on GA7G mRNA lacking downstream secondary structures.
- Provided the first experimental evidence for multiple frameshifting steps and -2 frameshifting.
- Confirmed the essentiality of EF-G·GTP, specifically GTP hydrolysis, for frameshifting.
- Showed that translation can occur in all three reading frames, induced by corresponding in-frame aminoacyl tRNAs.
Conclusions:
- GTP hydrolysis by EF-G·GTP is critical for codon-anticodon re-pairing during frameshifting.
- All three reading frames of a slippery sequence can be translated by cognate tRNAs, a novel observation.
- The developed assay method eliminates ambiguity from transcriptional slippage.
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