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De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data
Published on: February 18, 2022
FSscan: a mechanism-based program to identify +1 ribosomal frameshift hotspots
Pei-Yu Liao1, Yong Seok Choi, Kelvin H Lee
1School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York, USA.
Nucleic Acids Research
|September 29, 2009
Summary
A new bioinformatic tool, FSscan, systematically identifies +1 programmed ribosomal frameshifting (+1 PRF) sites in the Escherichia coli genome. This method aids in discovering novel frameshift proteins and understanding gene translation.
Area of Science:
- Computational Biology
- Molecular Biology
- Genomics
Background:
- Programmed ribosomal frameshifting (+1 PRF) is a translational control mechanism where ribosomes shift one nucleotide in the 3' direction.
- Existing methods for identifying +1 PRF sites rely on chance discovery or homology searches, limiting comprehensive analysis.
- Systematic identification of +1 PRF sites is crucial for understanding gene regulation and protein diversity.
Purpose of the Study:
- To develop and validate a computational framework, FSscan, for the systematic genome-wide identification of +1 PRF sites in Escherichia coli.
- To explore the potential of FSscan in discovering novel frameshift events and proteins within the E. coli genome.
Main Methods:
- Development of FSscan, a bioinformatic tool that scores potential +1 PRF sites based on known stimulatory signals and ribosome-tRNA interactions within a 16-nucleotide window.
- Application of FSscan to the entire Escherichia coli genome to identify candidate +1 PRF sites.
- In vivo experimental validation of predicted frameshift sites using reporter gene fusions and mass spectrometry analysis.
Main Results:
- FSscan successfully identified the known +1 PRF site in prfB and predicted five novel candidate sites, including yehP, pepP, nuoE, and cheA.
- Experimental validation confirmed significant +1 frameshifting activity at the predicted sites in vivo.
- Mass spectrometry analysis verified the expression of frameshifted proteins from a yehP-egfp fusion construct.
Conclusions:
- FSscan provides an effective genome-wide approach for the systematic discovery of +1 PRF sites in E. coli.
- The findings expand the known landscape of programmed ribosomal frameshifting and frameshifted proteins.
- This work has implications for identifying novel coding sequences, understanding translational regulation, and applying mass spectrometry in studying frameshifting events.
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