Engineering Fluorescently Labeled Ribosome for Observing mRNA-Ribosome Interactions at Single-Molecule Level
Date Issued
2016
Date
2016
Author(s)
Chang, You-Chiun
Abstract
Ribosomes are important machines for protein synthesis in all organisms. In the past 50 years, structures of the ribosome have been revealed by TEM, X-ray and cryo-EM. However, many aspects of the mechanism of how the ribosome translates mRNA into protein remain unclear. Here we aim to directly probe the interactions between the ribosome and mRNA by single-molecule Förster resonance energy transfer (smFRET). We choose to modify and fluorescently label the ribosomal protein S5 located at the mRNA entrance of the 30S subunit. We insert a ybbR-tag at the C terminus of the endogenous S5 protein (rpsE gene) by genomic replacement. The E. coli mutant grows normally without any apparent defects. Next, we use the SFP synthase to covalently attach Dy-547 or Dy-647 (equivalent to Cy3 or Cy5 respectively) to the ybbR-tag. Alternatively, the SNAP protein is inserted in place of the ybbR-tag, and the S5-SNAP fusion protein is overexpressed from a plasmid as an attempt to achieve better labeling efficiency with a much simpler procedure. After fluorescent labeling, we confirm the presence of the dye on the ribosome by SDS-PAGE and fluorescence imaging. The dye-labeled ribosome appears to be functional as indicated by electrophoretic mobility shift assay (EMSA) and in vitro translation with luciferase reporter assay. Single-molecule FRET signal can be observed from the initiation complex with a dye-labeled mRNA. Further optimization is needed for improvement of the labeling efficiency in order to effectively observe the interactions between the fluorescently labeled ribosome and mRNA.
Subjects
ribosome
single-molecule F?rster resonance energy transfer
ribosomal protein S5
rpsE gene
SNAP
Type
thesis
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